Patent application title: ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS FOR SEQUENCE MANIPULATION
Inventors:
Feng Zhang (Cambridge, MA, US)
Feng Zhang (Cambridge, MA, US)
Le Cong (Cambridge, MA, US)
Le Cong (Cambridge, MA, US)
IPC8 Class: AC12N1590FI
USPC Class:
Class name:
Publication date: 2015-08-20
Patent application number: 20150232882
Abstract:
The invention provides for systems, methods, and compositions for
manipulation of sequences and/or activities of target sequences. Provided
are vectors and vector systems, some of which encode one or more
components of a CRISPR complex, as well as methods for the design and use
of such vectors. Also provided are methods of directing CRISPR complex
formation in eukaryotic cells and methods for selecting specific cells by
introducing precise mutations utilizing the CRISPR-Cas system.Claims:
1. An engineered, non-naturally occurring Clustered Regularly
Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas)
(CRISPR-Cas) vector system comprising one or more vectors comprising: a)
a first regulatory element operably linked to one or more nucleotide
sequences encoding one or more CRISPR-Cas system polynucleotide sequences
comprising a guide sequence, a tracr RNA, and a tracr mate sequence,
wherein the guide sequence hybridizes with one or more target sequences
in polynucleotide loci in a eukaryotic cell, b) a second regulatory
element operably linked to a nucleotide sequence encoding a Type II Cas9
protein, wherein components (a) and (b) are located on same or different
vectors of the system, wherein the CRISPR-Cas system comprises two or
more nuclear localization signals (NLSs) expressed with the nucleotide
sequence encoding the Cas9 protein, whereby the one or more guide
sequences target the one or more polynucleotide loci in a eukaryotic cell
and the Cas9 protein cleaves the one or more polynucleotide loci, whereby
the sequence of the one or more polynucleotide loci is modified.
2. An engineered, non-naturally occurring Type II CRISPR-Cas vector system according to claim 1, wherein the Cas9 protein is mutated with respect to a corresponding wild type Cas9 protein such that the mutated protein is a nickase that lacks the ability to cleave one strand of a target polynucleotide, whereby the one or more guide sequences target the one or more polynucleotide loci in a eukaryotic cell and the Cas9 protein cleaves only one strand of the polynucleotide loci, whereby the sequence of the one or more polynucleotide loci is modified.
3. The system of claim 1 or 2, wherein the vectors are viral vectors.
4. The system of claim 3, wherein the viral vectors are retroviral, lentiviral, adenoviral, adeno-associated or herpes simplex viral vectors.
5. The system of any of claims 2-4 wherein the Cas9 protein comprises one or more mutations in the RuvC I, RuvC II or RuvC III catalytic domains.
6. The system of any of claims 2-4 wherein the Cas9 protein comprises a mutation selected from the group consisting of D10A, H840A, N854A and N863A with reference to the position numbering of a Streptococcus pyogenes Cas9 (SpCas9) protein.
7. The system of any preceding claim, wherein at least one NLS is at or near amino-terminus of the Cas9 protein and/or at least one NLS is at or near carboxy terminus of the Cas9 protein.
8. The system of claim 7, wherein at least one NLS is at or near amino-terminus of the Cas9 protein and at least one NLS is at or near carboxy terminus of the Cas9 protein.
9. The system of any preceding claim, wherein the one or more CRISPR-Cas system polynucleotide sequences comprise a guide sequence fused to a trans-activating cr (tracr) sequence.
10. The system of any preceding claim, wherein the CRISPR-Cas system polynucleotide sequence is a chimeric RNA comprising the guide sequence, the tracr sequence, and a tracr mate sequence.
11. The system of any preceding claim, wherein the eukaryotic cell is a mammalian cell or a human cell.
12. The system of any preceding claim, wherein the Cas9 protein is codon optimized for expression in a eukaryotic cell.
13. Use of the system of any of claims 1 to 12 for genome engineering, provided the use does not comprise a process for modifying the germ line genetic identity of human beings, and provided that said use is not a method for treatment of the human or animal body by surgery or therapy.
14. The use of claim 13 wherein the genome engineering comprises modifying a target polynucleotide in a eukaryotic cell, modifying expression of a polynucleotide in a eukaryotic cell, generating a model eukaryotic cell comprising a mutated disease gene, or knocking out a gene.
15. The use of claim 13 wherein the use further comprises repairing said cleaved target polynucleotide by inserting an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide.
16. The use of claim 13 wherein the use further comprises editing said cleaved target polynucleotide by inserting an exogenous template polynucleotide, wherein said edit results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide.
17. The use of claim 15 or 16 wherein the inserting is by homologous recombination.
18. Use of the system of any of claims 1 to 12 in the production of a non-human transgenic animal or transgenic plant.
Description:
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
[0001] This application in a continuation of International Application No. PCT/US2013/074819 filed Dec. 12, 2013, and published as PCT Publication No. WO 2014/093712 on Jun. 19, 2014 and which claims priority to U.S. provisional patent application 61/836,127 entitled ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED COMPOSITIONS FOR SEQUENCE MANIPULATION filed on Jun. 17, 2013. This application also claims priority to US provisional patent applications 61/758,468; 61/769,046; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130 each entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Jan. 30, 2013; Feb. 25, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013 respectively. Priority is also claimed to US provisional patent applications 61/736,527 and 61/748,427, both entitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec. 12, 2012 and Jan. 2, 2013, respectively. Priority is also claimed to US provisional patent applications 61/791,409 and 61/835,931 both entitled BI-2011/008/44790.02.2003 and BI-2011/008/44790.03.2003 filed on Mar. 15, 2013 and Jun. 17, 2013 respectively.
[0002] Reference is also made to US provisional patent applications 61/835,936, 61/836,101, 61/836,080, 61/836,123 and 61/835,973 each filed Jun. 17, 2013.
[0003] The foregoing applications, and all documents cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
FIELD OF THE INVENTION
[0005] The present invention generally relates to systems, methods and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that may use vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof.
BACKGROUND OF THE INVENTION
[0006] Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome.
SUMMARY OF THE INVENTION
[0007] There exists a pressing need for alternative and robust systems and techniques for sequence targeting with a wide array of applications. This invention addresses this need and provides related advantages. The CRISPR/Cas or the CRISPR-Cas system (both terms are used interchangeably throughout this application) does not require the generation of customized proteins to target specific sequences but rather a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target, in other words the Cas enzyme can be recruited to a specific DNA target using said short RNA molecule. Adding the CRISPR-Cas system to the repertoire of genome sequencing techniques and analysis methods may significantly simplify the methodology and accelerate the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. To utilize the CRISPR-Cas system effectively for genome editing without deleterious effects, it is critical to understand aspects of engineering and optimization of these genome engineering tools, which are aspects of the claimed invention.
[0008] In one aspect, the invention provides a vector system comprising one or more vectors. In some embodiments, the system comprises: (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a cell, e.g., eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence; wherein components (a) and (b) are located on the same or different vectors of the system. In some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system comprises the tracr sequence under the control of a third regulatory element, such as a polymerase III promoter. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, the CRISPR complex comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR complex in a detectable amount in the nucleus of a eukaryotic cell. Without wishing to be bound by theory, it is believed that a nuclear localization sequence is not necessary for CRISPR complex activity in eukaryotes, but that including such sequences enhances activity of the system, especially as to targeting nucleic acid molecules in the nucleus. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length. In general, and throughout this specification, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors." Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
[0009] Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
[0010] The term "regulatory element" is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF a promoter. Also encompassed by the term "regulatory element" are enhancer elements, such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit 3-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).
[0011] Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells.
[0012] In one aspect, the invention provides a vector comprising a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising one or more nuclear localization sequences. In some embodiments, said regulatory element drives transcription of the CRISPR enzyme in a eukaryotic cell such that said CRISPR enzyme accumulates in a detectable amount in the nucleus of the eukaryotic cell. In some embodiments, the regulatory element is a polymerase II promoter. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.
[0013] In one aspect, the invention provides a CRISPR enzyme comprising one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme lacks the ability to cleave one or more strands of a target sequence to which it binds.
[0014] In one aspect, the invention provides a eukaryotic host cell comprising (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence. In some embodiments, the host cell comprises components (a) and (b). In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell. In some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the eukaryotic host cell further comprises a third regulatory element, such as a polymerase III promoter, operably linked to said tracr sequence. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase 11 promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length. In an aspect, the invention provides a non-human eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments. In other aspects, the invention provides a eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments. The organism in some embodiments of these aspects may be an animal; for example a mammal. Also, the organism may be an arthropod such as an insect. The organism also may be a plant. Further, the organism may be a fungus.
[0015] In one aspect, the invention provides a kit comprising one or more of the components described herein. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence. In some embodiments, the kit comprises components (a) and (b) located on the same or different vectors of the system. In some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system further comprises a third regulatory element, such as a polymerase III promoter, operably linked to said tracr sequence. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length.
[0016] In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. In some embodiments, said cleavage comprises cleaving one or two strands at the location of the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in decreased transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cell, wherein the one or more vectors drive expression of one or more of: the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence. In some embodiments, said vectors are delivered to the eukaryotic cell in a subject. In some embodiments, said modifying takes place in said eukaryotic cell in a cell culture. In some embodiments, the method further comprises isolating said eukaryotic cell from a subject prior to said modifying. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject.
[0017] In one aspect, the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors drive expression of one or more of: the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence.
[0018] In one aspect, the invention provides a method of generating a model eukaryotic cell comprising a mutated disease gene. In some embodiments, a disease gene is any gene associated with an increase in the risk of having or developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotic cell, wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence; and (b) allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said disease gene, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence, thereby generating a model eukaryotic cell comprising a mutated disease gene. In some embodiments, said cleavage comprises cleaving one or two strands at the location of the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in decreased transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid changes in a protein expression from a gene comprising the target sequence.
[0019] In one aspect, the invention provides a method for developing a biologically active agent that modulates a cell signaling event associated with a disease gene. In some embodiments, a disease gene is any gene associated with an increase in the risk of having or developing a disease. In some embodiments, the method comprises (a) contacting a test compound with a model cell of any one of the described embodiments; and (b) detecting a change in a readout that is indicative of a reduction or an augmentation of a cell signaling event associated with said mutation in said disease gene, thereby developing said biologically active agent that modulates said cell signaling event associated with said disease gene.
[0020] In one aspect, the invention provides a recombinant polynucleotide comprising a guide sequence upstream of a tracr mate sequence, wherein the guide sequence when expressed directs sequence-specific binding of a CRISPR complex to a corresponding target sequence present in a eukaryotic cell. In some embodiments, the target sequence is a viral sequence present in a eukaryotic cell. In some embodiments, the target sequence is a proto-oncogene or an oncogene.
[0021] In one aspect the invention provides for a method of selecting one or more prokaryotic cell(s) by introducing one or more mutations in a gene in the one or more prokaryotic cell (s), the method comprising: introducing one or more vectors into the prokaryotic cell (s), wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and a editing template; wherein the editing template comprises the one or more mutations that abolish CRISPR enzyme cleavage; allowing homologous recombination of the editing template with the target polynucleotide in the cell(s) to be selected; allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence, wherein binding of the CRISPR complex to the target polynucleotide induces cell death, thereby allowing one or more prokaryotic cell(s) in which one or more mutations have been introduced to be selected. In a preferred embodiment, the CRISPR enzyme is Cas9. In another aspect of the invention the cell to be selected may be a eukaryotic cell. Aspects of the invention allow for selection of specific cells without requiring a selection marker or a two-step process that may include a counter-selection system.
[0022] In some aspects the invention provides a non-naturally occurring or engineered composition comprising a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence wherein (a), (b) and (c) are arranged in a 5' to 3' orientation, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
[0023] or
[0024] a CRISPR enzyme system, wherein the system is encoded by a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises (a) one or more guide sequences capable of hybridizing to one or more target sequences in a eukaryotic cell, (b) a tracr mate sequence, and (c) one or more tracr sequences, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences, wherein (a), (b) and (c) are arranged in a 5' to 3'orientation, wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, or a multiplexed CRISPR enzyme system, wherein the system is encoded by a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to (a) one or more guide sequences capable of hybridizing to a target sequence in a cell, and (b) at least one or more tracr mate sequences, II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and III. a third regulatory element operably linked to a tracr sequence, wherein components I, II and III are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and wherein in the multiplexed system multiple guide sequences and a single tracr sequence is used; and wherein one or more of the guide, tracr and tracr mate sequences are modified to improve stability.
[0025] In aspects of the invention, the modification comprises an engineered secondary structure. For example, the modification can comprise a reduction in a region of hybridization between the tracr mate sequence and the tracr sequence. For example, the modification also may comprise fusing the tracr mate sequence and the tracr sequence through an artificial loop. The modification may comprise the tracr sequence having a length between 40 and 120 bp. In embodiments of the invention, the tracr sequence is between 40 bp and full length of the tracr. In certain embodiments, the length of tracRNA includes at least nucleotides 1-67 and in some embodiments at least nucleotides 1-85 of the wild type tracRNA. In some embodiments, at least nucleotides corresponding to nucleotides 1-67 or 1-85 of wild type S. pyogenes Cas9 tracRNA may be used. Where the CRISPR system uses enzymes other than Cas9, or other than SpCas9, then corresponding nucleotides in the relevant wild type tracRNA may be present. In some embodiments, the length of tracRNA includes no more than nucleotides 1-67 or 1-85 of the wild type tracRNA. The modification may comprise sequence optimization. In certain aspects, sequence optimization may comprise reducing the incidence of polyT sequences in the tracr and/or tracr mate sequence. Sequence optimization may be combined with reduction in the region of hybridization between the tracr mate sequence and the tracr sequence; for example, a reduced length tracr sequence.
[0026] In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises reduction in polyT sequences in the tracr and/or tracr mate sequence. In some aspects of the invention, one or more Ts present in a poly-T sequence of the relevant wild type sequence (that is, a stretch of more than 3, 4, 5, 6, or more contiguous T bases; in some embodiments, a stretch of no more than 10, 9, 8, 7, 6 contiguous T bases) may be substituted with a non-T nucleotide, e.g., an A, so that the string is broken down into smaller stretches of Ts with each stretch having 4, or fewer than 4 (for example, 3 or 2) contiguous Ts. Bases other than A may be used for substitution, for example C or G, or non-naturally occurring nucleotides or modified nucleotides. If the string of Ts is involved in the formation of a hairpin (or stem loop), then it is advantageous that the complementary base for the non-T base be changed to complement the non-T nucleotide. For example, if the non-T base is an A, then its complement may be changed to a T, e.g., to preserve or assist in the preservation of secondary structure. For instance, 5'-TTTTT can be altered to become 5'-TTTAT and the complementary 5'-AAAAA can be changed into 5'-ATAAA.
[0027] In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises adding a polyT terminator sequence. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises adding a polyT terminator sequence in tracr and/or tracr mate sequences. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises adding a polyT terminator sequence in the guide sequence. The polyT terminator sequence may comprise 5 contiguous T bases, or more than 5.
[0028] In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises altering loops and/or hairpins. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises providing a minimum of two hairpins in the guide sequence. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises providing a hairpin formed by complementation between the tracr and tracr mate (direct repeat) sequence. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises providing one or more further hairpin(s) at or towards the 3' end of the tracrRNA sequence. For example, a hairpin may be formed by providing self complementary sequences within the tracRNA sequence joined by a loop such that a hairpin is formed on self folding. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises providing additional hairpins added to the 3' of the guide sequence. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises extending the 5' end of the guide sequence. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises providing one or more hairpins in the 5' end of the guide sequence. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises appending the sequence (5'-AGGACGAAGTCCTAA) to the 5' end of the guide sequence. Other sequences suitable for forming hairpins will be known to the skilled person, and may be used in certain aspects of the invention. In some aspects of the invention, at least 2, 3, 4, 5, or more additional hairpins are provided. In some aspects of the invention, no more than 10, 9, 8, 7, 6 additional hairpins are provided. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises two hairpins. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises three hairpins. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises at most five hairpins.
[0029] In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises providing cross linking, or providing one or more modified nucleotides in the polynucleotide sequence. Modified nucleotides and/or cross linking may be provided in any or all of the tracr, tracr mate, and/or guide sequences, and/or in the enzyme coding sequence, and/or in vector sequences. Modifications may include inclusion of at least one non naturally occurring nucleotide, or a modified nucleotide, or analogs thereof. Modified nucleotides may be modified at the ribose, phosphate, and/or base moiety. Modified nucleotides may include 2'-O-methyl analogs, 2'-deoxy analogs, or 2'-fluoro analogs. The nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used. The use of locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be possible. Further examples of modified bases include, but are not limited to. 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine.
[0030] It will be understood that any or all of the above modifications may be provided in isolation or in combination in a given CRISPR-Cas system or CRISPR enzyme system. Such a system may include one, two, three, four, five, or more of said modifications.
[0031] In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the CRISPR enzyme is a type II CRISPR system enzyme, e.g., a Cas9 enzyme. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the CRISPR enzyme is comprised of less than one thousand amino acids, or less than four thousand amino acids. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the Cas9 enzyme is StCas9 or St1Cas9, or the Cas9 enzyme is a Cas9 enzyme from an organism selected from the group consisting of genus Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium or Corynebacter. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the CRISPR enzyme is a nuclease directing cleavage of both strands at the location of the target sequence.
[0032] In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the first regulatory element is a polymerase III promoter. In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the second regulatory element is a polymerase II promoter.
[0033] In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the guide sequence comprises at least fifteen nucleotides.
[0034] In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the modification comprises optimized tracr sequence and/or optimized guide sequence RNA and/or co-fold structure of tracr sequence and/or tracr mate sequence(s) and/or stabilizing secondary structures of tracr sequence and/or tracr sequence with a reduced region of base-pairing and/or tracr sequence fused RNA elements; and/or, in the multiplexed system there are two RNAs comprising a tracer and comprising a plurality of guides or one RNA comprising a plurality of chimerics.
[0035] In aspects of the invention the chimeric RNA architecture is further optimized according to the results of mutagenesis studies. In chimeric RNA with two or more hairpins, mutations in the proximal direct repeat to stabilize the hairpin may result in ablation of CRISPR complex activity. Mutations in the distal direct repeat to shorten or stabilize the hairpin may have no effect on CRISPR complex activity. Sequence randomization in the bulge region between the proximal and distal repeats may significantly reduce CRISPR complex activity. Single base pair changes or sequence randomization in the linker region between hairpins may result in complete loss of CRISPR complex activity. Hairpin stabilization of the distal hairpins that follow the first hairpin after the guide sequence may result in maintenance or improvement of CRISPR complex activity. Accordingly, in preferred embodiments of the invention, the chimeric RNA architecture may be further optimized by generating a smaller chimeric RNA which may be beneficial for therapeutic delivery options and other uses and this may be achieved by altering the distal direct repeat so as to shorten or stabilize the hairpin. In further preferred embodiments of the invention, the chimeric RNA architecture may be further optimized by stabilizing one or more of the distal hairpins. Stabilization of hairpins may include modifying sequences suitable for forming hairpins. In some aspects of the invention, at least 2, 3, 4, 5, or more additional hairpins are provided. In some aspects of the invention, no more than 10, 9, 8, 7, 6 additional hairpins are provided. In some aspects of the invention stabilization may be cross linking and other modifications. Modifications may include inclusion of at least one non naturally occurring nucleotide, or a modified nucleotide, or analogs thereof. Modified nucleotides may be modified at the ribose, phosphate, and/or base moiety. Modified nucleotides may include 2'-O-methyl analogs, 2'-deoxy analogs, or 2'-fluoro analogs. The nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used. The use of locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be possible. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine.
[0036] In an aspect the invention provides the CRISPR-Cas system or CRISPR enzyme system wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.
[0037] Accordingly, in some aspects of the invention, the length of tracRNA required in a construct of the invention, e.g., a chimeric construct, need not necessarily be fixed, and in some aspects of the invention it can be between 40 and 120 bp, and in some aspects of the invention up to the full length of the tracr, e.g., in some aspects of the invention, until the 3' end of tracr as punctuated by the transcription termination signal in the bacterial genome. In certain embodiments, the length of tracRNA includes at least nucleotides 1-67 and in some embodiments at least nucleotides 1-85 of the wild type tracRNA. In some embodiments, at least nucleotides corresponding to nucleotides 1-67 or 1-85 of wild type S. pyogenes Cas9 tracRNA may be used. Where the CRISPR system uses enzymes other than Cas9, or other than SpCas9, then corresponding nucleotides in the relevant wild type tracRNA may be present. In some embodiments, the length of tracRNA includes no more than nucleotides 1-67 or 1-85 of the wild type tracRNA With respect to sequence optimization (e.g., reduction in polyT sequences), e.g., as to strings of Ts internal to the tracr mate (direct repeat) or tracrRNA, in some aspects of the invention, one or more Ts present in a poly-T sequence of the relevant wild type sequence (that is, a stretch of more than 3, 4, 5, 6, or more contiguous T bases; in some embodiments, a stretch of no more than 10, 9, 8, 7, 6 contiguous T bases) may be substituted with a non-T nucleotide, e.g., an A, so that the string is broken down into smaller stretches of Ts with each stretch having 4, or fewer than 4 (for example, 3 or 2) contiguous Ts. If the string of Ts is involved in the formation of a hairpin (or stem loop), then it is advantageous that the complementary base for the non-T base be changed to complement the non-T nucleotide. For example, if the non-T base is an A, then its complement may be changed to a T, e.g., to preserve or assist in the preservation of secondary structure. For instance, 5'-TTTTT can be altered to become 5'-TTTAT and the complementary 5'-AAAAA can be changed into 5'-ATAAA. As to the presence of polyT terminator sequences in tracr+tracr mate transcript, e.g., a polyT terminator (TTTTT or more), in some aspects of the invention it is advantageous that such be added to end of the transcript, whether it is in two RNA (tracr and tracr mate) or single guide RNA form. Concerning loops and hairpins in tracr and tracr mate transcripts, in some aspects of the invention it is advantageous that a minimum of two hairpins be present in the chimeric guide RNA. A first hairpin can be the hairpin formed by complementation between the tracr and tracr mate (direct repeat) sequence. A second hairpin can be at the 3' end of the tracrRNA sequence, and this can provide secondary structure for interaction with Cas9. Additional hairpins may be added to the 3' of the guide RNA, e.g., in some aspects of the invention to increase the stability of the guide RNA. Additionally, the 5' end of the guide RNA, in some aspects of the invention, may be extended. In some aspects of the invention, one may consider 20 bp in the 5' end as a guide sequence. The 5' portion may be extended. One or more hairpins can be provided in the 5' portion, e.g., in some aspects of the invention, this may also improve the stability of the guide RNA. In some aspects of the invention, the specific hairpin can be provided by appending the sequence (5'-AGGACGAAGTCCTAA) to the 5' end of the guide sequence, and, in some aspects of the invention, this may help improve stability. Other sequences suitable for forming hairpins will be known to the skilled person, and may be used in certain aspects of the invention. In some aspects of the invention, at least 2, 3, 4, 5, or more additional hairpins are provided. In some aspects of the invention, no more than 10, 9, 8, 7, 6 additional hairpins are provided. The foregoing also provides aspects of the invention involving secondary structure in guide sequences. In some aspects of the invention there may be cross linking and other modifications, e.g., to improve stability. Modifications may include inclusion of at least one non naturally occurring nucleotide, or a modified nucleotide, or analogs thereof. Modified nucleotides may be modified at the ribose, phosphate, and/or base moiety. Modified nucleotides may include 2'-O-methyl analogs, 2'-deoxy analogs, or 2'-fluoro analogs. The nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used. The use of locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be possible. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. Such modifications or cross linking may be present in the guide sequence or other sequences adjacent the guide sequence.
[0038] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.
[0039] It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included". "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0041] FIG. 1 shows a schematic model of the CRISPR system. The Cas9 nuclease from Streptococcus pyogenes (yellow) is targeted to genomic DNA by a synthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue) and a scaffold (red). The guide sequence base-pairs with the DNA target (blue), directly upstream of a requisite 5'-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB) ˜3 bp upstream of the PAM (red triangle).
[0042] FIG. 2A-F illustrates an exemplary CRISPR system, a possible mechanism of action, an example adaptation for expression in eukaryotic cells, and results of tests assessing nuclear localization and CRISPR activity.
[0043] FIG. 3A-C illustrates an exemplary expression cassette for expression of CRISPR system elements in eukaryotic cells, predicted structures of example guide sequences, and CRISPR system activity as measured in eukaryotic and prokaryotic cells.
[0044] FIG. 4A-D illustrates results of an evaluation of SpCas9 specificity for an example target.
[0045] FIG. 5A-G illustrates an exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells.
[0046] FIG. 6A-C illustrates a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting.
[0047] FIG. 7A-D illustrates an exemplary CRISPR system, an example adaptation for expression in eukaryotic cells, and results of tests assessing CRISPR activity.
[0048] FIG. 8A-C illustrates exemplary manipulations of a CRISPR system for targeting of genomic loci in mammalian cells.
[0049] FIG. 9A-B illustrates the results of a Northern blot analysis of crRNA processing in mammalian cells.
[0050] FIG. 10A-C illustrates a schematic representation of chimeric RNAs and results of SURVEYOR assays for CRISPR system activity in eukaryotic cells.
[0051] FIG. 11A-B illustrates a graphical representation of the results of SURVEYOR assays for CRISPR system activity in eukaryotic cells.
[0052] FIG. 12 illustrates predicted secondary structures for exemplary chimeric RNAs comprising a guide sequence, tracr mate sequence, and tracr sequence.
[0053] FIG. 13A-D is a phylogenetic tree of Cas genes
[0054] FIG. 14A-F shows the phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (˜1400 amino acids) and two of small Cas9s (˜1100 amino acids).
[0055] FIG. 15 shows a graph depicting the function of different optimized guide RNAs.
[0056] FIG. 16 shows the sequence and structure of different guide chimeric RNAs.
[0057] FIG. 17 shows the co-fold structure of the tracrRNA and direct repeat.
[0058] FIGS. 18 A and B shows data from the StlCas9 chimeric guide RNA optimization in vitro.
[0059] FIG. 19A-B shows cleavage of either unmethylated or methylated targets by SpCas9 cell lysate.
[0060] FIG. 20A-G shows the optimization of guide RNA architecture for SpCas9-mediated mammalian genome editing. (a) Schematic of bicistronic expression vector (PX330) for U6 promoter-driven single guide RNA (sgRNA) and CBh promoter-driven human codon-optimized Streptococcus pyogenes Cas9 (hSpCas9) used for all subsequent experiments. The sgRNA consists of a 20-nt guide sequence (blue) and scaffold (red), truncated at various positions as indicated. (b) SURVEYOR assay for SpCas9-mediated indels at the human EMX1 and PVALB loci. Arrows indicate the expected SURVEYOR fragments (n=3). (c) Northern blot analysis for the four sgRNA truncation architectures, with U1 as loading control. (d) Both wildtype (wt) or nickase mutant (D10A) of SpCas9 promoted insertion of a HindIII site into the human EMX1 gene. Single stranded oligonucleotides (ssODNs), oriented in either the sense or antisense direction relative to genome sequence, were used as homologous recombination templates. (e) Schematic of the human SERPINB5 locus. sgRNAs and PAMs are indicated by colored bars above sequence; methylcytosine (Me) are highlighted (pink) and numbered relative to the transcriptional start site (TSS, +1). (f) Methylation status of SERPINB5 assayed by bisulfite sequencing of 16 clones. Filled circles, methylated CpG; open circles, unmethylated CpG. (g) Modification efficiency by three sgRNAs targeting the methylated region of SERPINB5, assayed by deep sequencing (n=2). Error bars indicate Wilson intervals (Online Methods).
[0061] FIG. 21A-B shows the further optimization of CRISPR-Cas sgRNA architecture. (a) Schematic of four additional sgRNA architectures, I-IV. Each consists of a 20-nt guide sequence (blue) joined to the direct repeat (DR, grey), which hybridizes to the tracrRNA (red). The DR-tracrRNA hybrid is truncated at +12 or +22, as indicated, with an artificial GAAA stem loop. tracrRNA truncation positions are numbered according to the previously reported transcription start site for tracrRNA. sgRNA architectures II and IV carry mutations within their poly-U tracts, which could serve as premature transcriptional terminators. (b) SURVEYOR assay for SpCas9-mediated indels at the human EMX1 locus for target sites 1-3. Arrows indicate the expected SURVEYOR fragments (n=3).
[0062] FIG. 22 illustrates visualization of some target sites in the human genome.
[0063] FIG. 23A-B shows (A) a schematic of the sgRNA and (B) the SURVEYOR analysis of five sgRNA variants for SaCas9 for an optimal truncated architecture with highest cleavage efficiency
[0064] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.
DETAILED DESCRIPTION OF THE INVENTION
[0065] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
[0066] In aspects of the invention the terms "chimeric RNA", "chimeric guide RNA", "guide RNA", "single guide RNA" and "synthetic guide RNA" are used interchangeably and refer to the polynucleotide sequence comprising the guide sequence, the tracr sequence and the tracr mate sequence. The term "guide sequence" refers to the about 20 bp sequence within the guide RNA that specifies the target site and may be used interchangeably with the terms "guide" or "spacer". The term "tracr mate sequence" may also be used interchangeably with the term "direct repeat(s)".
[0067] As used herein the term "wild type" is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
[0068] As used herein the term "variant" should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature.
[0069] The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
[0070] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base-pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
[0071] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, N.Y.
[0072] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the "complement" of the given sequence.
[0073] As used herein, "stabilization" or "increasing stability" with respect to components of the CRISPR system relate to securing or steadying the structure of the molecule. This may be accomplished by introduction of one or mutations, including single or multiple base pair changes, increasing the number of hair pins, cross linking, breaking up particular stretches of nucleotides and other modifications. Modifications may include inclusion of at least one non naturally occurring nucleotide, or a modified nucleotide, or analogs thereof. Modified nucleotides may be modified at the ribose, phosphate, and/or base moiety. Modified nucleotides may include 2'-O-methyl analogs, 2'-deoxy analogs, or 2'-fluoro analogs. The nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used. The use of locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be possible. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. These modifications may apply to any component of the CRSIPR system. In a preferred embodiment these modifications are made to the RNA components, e.g. the guide RNA or chimeric polynucleotide sequence.
[0074] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
[0075] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
[0076] The terms "subject." "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. In some embodiments, a subject may be an invertebrate animal, for example, an insect or a nematode; while in others, a subject may be a plant or a fungus.
[0077] The terms "therapeutic agent", "therapeutic capable agent" or "treatment agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
[0078] As used herein, "treatment" or "treating," or "palliating" or "ameliorating" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
[0079] The term "effective amount" or "therapeutically effective amount" refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
[0080] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).
[0081] Several aspects of the invention relate to vector systems comprising one or more vectors, or vectors as such. Vectors can be designed for expression of CRISPR transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press. San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
[0082] Vectors may be introduced and propagated in a prokaryote. In some embodiments, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system). In some embodiments, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
[0083] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
[0084] In some embodiments, a vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
[0085] In some embodiments, a vector drives protein expression in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
[0086] In some embodiments, a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0087] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
[0088] In some embodiments, a regulatory element is operably linked to one or more elements of a CRISPR system so as to drive expression of the one or more elements of the CRISPR system. In general, CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDRs (SPacer Interspersed Direct Repeats), constitute a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433
[1987]; and Nakata et al., J. Bacteriol., 171:3553-3556
[1989]), and associated genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacteriumn tuberculosis (See, Groenen et al., Mol. Microbiol., 10:1057-1065
[1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263
[1999]; Masepohl et al., Biochim. Biophys. Acta 1307:26-30
[1996]; and Mojica et al., Mol. Microbiol., 17:85-93
[1995]). The CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33
[2002]; and Mojica et al., Mol. Microbiol., 36:244-246
[2000]). In general, the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al.,
[2000], supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401
[2000]). CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575
[2002]; and Mojica et al.,
[2005]) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.
[0089] In general, "CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridisation and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast. A sequence or template that may be used for recombination into the the targeted locus comprising the target sequences is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In aspects of the invention, an exogenous template polynucleotide may be referred to as an editing template. In an aspect of the invention the recombination is homologous recombination.
[0090] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridise and participate in formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity is not needed, provided there is sufficient to be functional. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.
[0091] In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site"). In some embodiments, one or more insertion sites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and/or downstream of one or more sequence elements of one or more vectors. In some embodiments, a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.
[0092] In some embodiments, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, CasB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments the CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A. In some embodiments, a Cas9 nickase may be used in combination with guide sequenc(es), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ. Applicants have demonstrated (data not shown) the efficacy of two nickase targets (i.e., sgRNAs targeted at the same location but to different strands of DNA) in inducing mutagenic NHEJ. A single nickase (Cas9-D10A with a single sgRNA) is unable to induce NHEJ and create indels but Applicants have shown that double nickase (Cas9-D10A and two sgRNAs targeted to different strands at the same location) can do so in human embryonic stem cells (hESCs). The efficiency is about 50% of nuclease (i.e., regular Cas9 without D10 mutation) in hESCs.
[0093] As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity. In some embodiments, a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with respect to its non-mutated form. Other mutations may be useful; where the Cas9 or other CRISPR enzyme is from a species other than S. pyogenes, mutations in corresponding amino acids may be made to achieve similar effects.
[0094] In some embodiments, an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database", and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.
[0095] In some embodiments, a vector encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In a preferred embodiment of the invention, the CRISPR enzyme comprises at most 6 NLSs. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Typically, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are known. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV; the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain from importin-alpha; the sequences VSRKRPRP and PPKKARED of the myoma T protein; the sequence POPKKKPL of human p53; the sequence SALIKKKKKMAP of mouse c-ab1 IV; the sequences DRLRR and PKQKKRK of the influenza virus NS1; the sequence RKLKKKIKKL of the Hepatitis virus delta antigen; the sequence REKKKFLKRR of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK of the steroid hormone receptors (human) glucocorticoid.
[0096] In general, the one or more NLSs are of sufficient strength to drive accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the CRISPR enzyme, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the CRISPR enzyme, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI). Examples of detectable markers include fluorescent proteins (such as Green fluorescent proteins, or GFP; RFP; CFP), and epitope tags (HA tag, flag tag, SNAP tag). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g. assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity), as compared to a control no exposed to the CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the one or more NLSs.
[0097] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.
[0098] A guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG where NNNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG where NNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. For the S. thermophilus CRISPR1 Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW where NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. A unique target sequence in a genome may include an S. thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW where NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. For the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG where NNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. In each of these sequences "M" may be A, G, T, or C, and need not be considered in identifying a sequence as unique.
[0099] In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62). Further algorithms may be found in U.S. application Ser. No. TBA (Broad Reference BI-2012/084 44790.11.2022); incorporated herein by reference.
[0100] In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. Example illustrations of optimal alignment between a tracr sequence and a tracr mate sequence are provided in FIGS. 12B and 13B. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. Preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. The sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. In an embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the invention, the transcript has at most five hairpins. In some embodiments, the single transcript further includes a transcription termination sequence; preferably this is a polyT sequence, for example six T nucleotides. An example illustration of such a hairpin structure is provided in the lower portion of FIG. 13B, where the portion of the sequence 5' of the final "N" and upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3' of the loop corresponds to the tracr sequence. Further non-limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5' to 3'), where "N" represents a base of a guide sequence, the first block of lower case letters represent the tracr mate sequence, and the second block of lower case letters represent the tracr sequence, and the final poly-T sequence represents the transcription terminator: (1) NNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggctt catgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT; (2) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaa- atca acaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT; (3) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaa- atca acaccctgtcattttatggcagggtgtTTTTTT; (4) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttg- aaaa agtggcaccgagtcggtgcTTTTTT; (5) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttg- aa aaagtgTTTTTT; and (6) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTT TTT. In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence (such as illustrated in the top portion of FIG. 13B).
[0101] In some embodiments, a recombination template is also provided. A recombination template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex. A template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence. When optimally aligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.
[0102] In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GALA DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.
[0103] In some aspects, the invention provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to a cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt. Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).
[0104] Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
[0105] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
[0106] The use of RNA or DNA viral based systems for the delivery of nucleic acids takes advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
[0107] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700).
[0108] In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
[0109] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
[0110] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7. HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, T1B55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr-/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-SF, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a CRISPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.
[0111] In some embodiments, one or more vectors described herein are used to produce a non-human transgenic animal or transgenic plant. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, or rabbit. In certain embodiments, the organism or subject is a plant. In certain embodiments, the organism or subject or plant is algae. Methods for producing transgenic plants and animals are known in the art, and generally begin with a method of cell transfection, such as described herein. Transgenic animals are also provided, as are transgenic plants, especially crops and algae. The transgenic animal or plant may be useful in applications outside of providing a disease model. These may include food or feed production through expression of, for instance, higher protein, carbohydrate, nutrient or vitamins levels than would normally be seen in the wildtype. In this regard, transgenic plants, especially pulses and tubers, and animals, especially mammals such as livestock (cows, sheep, goats and pigs), but also poultry and edible insects, are preferred.
[0112] Transgenic algae or other plants such as rape may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol), for instance. These may be engineered to express or overexpress high levels of oil or alcohols for use in the oil or biofuel industries.
[0113] In one aspect, the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
[0114] In one aspect, the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
[0115] With recent advances in crop genomics, the ability to use CRISPR-Cas systems to perform efficient and cost effective gene editing and manipulation will allow the rapid selection and comparison of single and and multiplexed genetic manipulations to transform such genomes for improved production and enhanced traits. In this regard reference is made to US patents and publications: U.S. Pat. No. 6,603,061--Agrobacterium-Mediated Plant Transformation Method; U.S. Pat. No. 7,868,149--Plant Genome Sequences and Uses Thereof and US 2009/0100536--Transgenic Plants with Enhanced Agronomic Traits, all the contents and disclosure of each of which are herein incorporated by reference in their entirety. In the practice of the invention, the contents and disclosure of Morrell et al "Crop genomics:advances and applications" Nat Rev Genet. 2011 Dec. 29; 13(2):85-96 are also herein incorporated by reference in their entirety. In an advantageous embodiment of the invention, the CRISPR/Cas9 system is used to engineer microalgae (Example 14). Accordingly, reference herein to animal cells may also apply, mutatis mutandis, to plant cells unless otherwise apparent.
[0116] In one aspect, the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including micro-algae), and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant (including micro-algae).
[0117] In one aspect, the invention provides kits containing any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting a guide sequence upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence. Elements may provide individually or in combinations, and may provided in any suitable container, such as a vial, a bottle, or a tube. In some embodiments, the kit includes instructions in one or more languages, for example in more than one language.
[0118] In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element. In some embodiments, the kit comprises a homologous recombination template polynucleotide.
[0119] In one aspect, the invention provides methods for using one or more elements of a CRISPR system. The CRISPR complex of the invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types. As such the CRISPR complex of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.
[0120] The target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell. The target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA). Without wishing to be bound by theory, it is believed that the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognised by the CRISPR complex. The precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence) Examples of PAM sequences are given in the examples section below, and the skilled person will be able to identify further PAM sequences for use with a given CRISPR enzyme.
[0121] The target polynucleotide of a CRISPR complex may include a number of disease-associated genes and polynucleotides as well as signaling biochemical pathway-associated genes and polynucleotides as listed in US provisional patent applications 61/736,527 and 61/748,427 having Broad reference BI-2011/008/WSGR Docket No. 44063-701.101and BI-2011/008/WSGR Docket No. 44063-701.102 respectively, both entitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec. 12, 2012 and Jan. 2, 2013, respectively, the contents of all of which are herein incorporated by reference in their entirety.
[0122] Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A "disease-associated" gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at a normal or abnormal level.
[0123] Examples of disease-associated genes and polynucleotides are available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web.
[0124] Examples of disease-associated genes and polynucleotides are listed in Tables A and B. Disease specific information is available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web. Examples of signaling biochemical pathway-associated genes and polynucleotides are listed in Table C.
[0125] Mutations in these genes and pathways can result in production of improper proteins or proteins in improper amounts which affect function. Further examples of genes, diseases and proteins are hereby incorporated by reference from US Provisional applications 61/736,527 and 61/748,427. Such genes, proteins and pathways may be the target polynucleotide of a CRISPR complex.
TABLE-US-00001 TABLE A DISEASE/DISORDER GENE(S) Neoplasia PTEN; ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; AKT; AKT2; .AKT3; HIF; HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1 (Wilms Tumor); FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB (retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR (Androgen Receptor); TSG101; IGF; IGF Receptor; Igfl (4 variants); Igf2 (3 variants); Igf 1 Receptor; Igf 2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related Abcr; Ccl2.; Cc2; cp (ceruloplasmin); Macular Timp3; cathepsinD; Degeneration Vldlrr; Ccr2 Schizophrenia Neuregulin 1 (Nrg 1); Erb4 Disorders (receptor for Neuregulin); Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2 Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a; 5-HTT (slc6a4); COMT; DRD (Drd1a); SLC6A3 DTNBP1; Dao (Dao1) Trinucleotide HTT (Huntington's Dx); Repeat SBMA/SMAX1/AR (Kennedy's Disorders Dx); FXN/X25 (Friedrich's Ataxia); ATX3 (Machado- Joseph's Dx); ATXN1, and ATXN2 (spinocerebellar ataxias); I)MPK (myotonic dystrophy); Atrophin-1 and Atn1 (DRPLA Dx); CBP (Creb-BP- global instability); VLDLR (Alzheimer's); Atxn7; Atxn10 Fragile X Syndrome FMR2; FXR1; FXR2; mGLUR5 Secretase Related APH-1 (alpha and beta): Disorders Presenilin (Psen1): nicastrin (Ncstn); PEN-2 Others Nos1; Parp1; Nat 1; Nat2. Prion-related disorders Prp ALS SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c) Drug addiction Prkce (alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2; Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Grial (alcohol) Autism Mecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1; Fragile X (FMR2 (AFF2); FXR1; FXR2; Mglur5) Alzheimer`s Disease E1; CHIP; UGH; UBB; Tau; LRP; PICALM; Clusterin; PS1; SORL1; CR1; V1dlr; Ubal; Uba3; CHIP28 (Aqp1, Aquaporin 1); Uchl1; Uchl3; APP Inflammation IL-10;1L-1 (IL-1a; IL-1b); 1L-13; 1L-17 (1L-17a (CTLA8); IL- 17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1; ptpn22; TNFa; NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4; Cx3cl1 Parkinson`s Disease x-Synuclein; DJ-1; LRRK2; Parkin; PINK1
TABLE-US-00002 TABLE B Blood and Anemia (CDAN1, CDAI, RPS19, DBA, PKLR, PKI, coagulation NT5C3, UMPHl, diseases PSN1, RHAG, RH50A., NRAMP2, SBTP, and ALAS2, ANH1, ASB, disorders ABCB7, ABC7, ASAT); Bare lymphocyte syndrome (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5), Bleeding disorders (TBXA2R, P2RX1, P2X1); Factor H and factor H-like 1 (HF1, CFH, HUS); Factor V and factor VIII (MCFD2); Factor VII deficiency (F7); Factor X deficiency (F10); Factor XI deficiency (F11); Factor XII deficiency (F12, HAF); Factor XIIIA deficiency (F13A1, F13A); Factor XIIIB deficiency (F13B); Fanconi anemia (FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FULJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1, BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); Hemophagocytic lymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B (F9, HEMB), Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficiencies and disorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, E1F2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia (HBA2, HBB, HBD, LCRB, HBA1). Cell B-cell non-Hodgkin lymphoma (BCL7A dysregulation BCL7); Leukemia (TAL1, and TCL5, SCL, TAL2, FLT3, oncology NBS1, NBS, ZNFN1A1, IK1, LYF1, diseases HOXD4, HOX4B; BCR, CML, PHL, and ALL, ARNT, KRAS2, RASK2, disorders GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AMLI, WHSC1L1, NSD3, FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN), Inflammation AIDS (KIR3DL1, NKAT3, NKB1, AMB11, and KIR3DS1, IFNG, CXCL12, immune SDF1); Autoimmune lymphoproliferative syndrome related (TNFRSF6, APT1, diseases FAS, CD95, ALPS1A); Combined and immunodeficiency, (IL2RG, disorders SCIDX1, SCIDX, 1MD4); HIV-1 (CCL5, SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5. CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI; Inflammation (IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f), IL-23, Cx3crl, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3l1); Severe combined immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDXI, SCIDX, IMD4). Metabolic, Amyloid neuropathy (TTR, PALB); liver, Amyloidosis (APOA1, APP, AAA, kidney CVAP, AD1, GSN, FGA, LYZ, TTR, PALB); and protein Cirrhosis (KRT18, KRT8, diseases CIRHIA, NAIC, TEX292, KIAA1988); and Cystic fibrosis (CFTR, ABCC7, disorders CF, MRP7); Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1, HNF1A, MODY3), Hepatic failure, early onset, and neurologic disorder (SCOD1, SCO1), Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancer and carcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; Medullary cystic kidney disease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1, QDPR, DHPR, PTS); Polycystic kidney and hepatic disease (FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63). Muscular/ Becker muscular dystrophy (DMD, Skeletal BMD, MYF6), Duchenne Muscular diseases Dystrophy (DMD, BMD); and Emery-Dreifuss muscular dystrophy disorders (LMNA,LMN I, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A); Facioscapulohumeral muscular dystrophy (FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SOCA, ADL, DAG2, LGM )2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7, OC116, OPTB1); Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1). Neurological ALS (SOD1, ALS2, STEX, FUS, TARDBP, and VEGF (VEGF-a, VEGF-b,VEGF-c); Alzheimer neuronal disease (APP, AAA, CVAP, AD1, APOE, AD2, diseases PSEN2, AD4, STM2, and APBB2, FE65L1, NOS3, PLAU, URK, ACE, disorders DCP1, ACE1, MPO, PACIP1 PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5); Huntington's disease and disease like disorders (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP; PARK1, PARK4, DJ1, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH, NDUF-V2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, , STK9, MECP2, RTT, PPMX, MRXI6 MRX79, x-Synuclein, DJ-1); Schizophrenia (Neuregulin1 (Nrg1) Erb4 (receptor for Neuregulin), Complexin1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (S1c6a4), COW, DRD (Drd la), SLC6A3, DAOA, DTNBP1, Dao (Dao1)); Secretase Related Disorders (APH-1 (alpha and beta), Presenitin (Pseni), nicastrin, (Ncstn), PEN-2, Nos1, Parp1, Nat1, Nat2); Trinucleotide Repeat Disorders (HTT (Huntington's Dx), SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich's Ataxia), ATX3 (Machado- Joseph's Dx), ATXN1 and ATXN2 (spinocerebellar ataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP (Creb-BP- global instability), VLDLR (Alzheimer's), Atxn7, Atxn10). Occular Age-related macular degeneration diseases (Abcr, Ccl2, Cc2, cp (ceruloplasmin), and Timp3, cathepsinD, Vldlr, Ccr2); disorders Cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYAI, GJA8, CX5O, CAEI , CJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1); Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGGI, CSD, BIGH3, CDG2, TACSTD2, TROP2, MIS1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma (MYOC, TIGR, GLCIA, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1 NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1, RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP2O, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).
TABLE-US-00003 TABLE C CELLULAR FUNCTION GENES PI3K/AKT PRKCE.; ITGAM; ITGA5; Signaling IRAK1; PRKAA2; EfF2AK2; PTEN; EIF4E; PRKCZ; GRK6; MAPK1; TSC1; PLK1; AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8; BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1; MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB; DYRK1A; CDKNJA; ITGB1; MAP2K2; JAM; AKTI; JAK2; PIK3RI; CHUK; PDPK1; PPP2R5C; CTNNB1; MAP2KI; NFKBI; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN; ITGA2; TTK.; CSNK1A1 ; BRAF; GSK3B; AKT3; FOXO1; SGK.; HSP90AA.1; RPS6KB1 ERK/MAPK PRKCE; ITGAM; ITGA5; Signaling HSPB1; IRAKI; PRKAA2; EIF2AK2; RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPKI; RAC2; PLK1; AKT2; PIK3CA; CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8; MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1.; PPARG; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PPP2CA; PIMI; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSRI; PXN; RAF1; FYN; DYRK1A; ITGB1 MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3; ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF; STAT1; SGK Glucocorticoid RAC1; TAF4B; EP300; SMAD2; Receptor TRAF6; PCAF; ELKI; Signaling MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA; CREBI; FOS; HSPA5; NFKB ; BCL2; MAP3K.14; STAT5B; PIK3C9; PIK3C3; MAPK8; BCL2L1; MAPK3; TSC22D3; MAPK10; NRIPI; KRAS; MAPK13; RELA.; STAT5A; MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3; MAPK 14; TNF; RAF1; .IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8; NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBRl; ESR1; SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 Axonal PRKCE; ITGAM; ROCK1; Guidance ITGA5; CXCR4; ADAM12; Signaling IGFI; RACI; RAP1A; E1F4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ; PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABLI; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA Ephrin PRKCE; ITGAM; ROCK1; Receptor ITGA5; CXCR4; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1; AKT2; DOKI; CDK8; CREB1.; PTK2; CFL1.; GNAQ; MAP3K14; CXCL12;MAPK8; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4; AKT1; JAK2; STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK; CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK Actin ACTN4; PRKCE; ITGAM; ROCK1; Cytoskeleton ITGA5; IRAKI; Signaling PRKAA2; EIF2AK2; RAC1; INS; ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1 ; P1K3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN; VIL2; RAF1; GSN; DYRKIA; ITGB1; MAP2K2; PAK4; P1P5K1A; PIK3R1; MAP2K1; PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGK Huntington`s PRKCE; IGF1; EP300; RCOR1; Disease PRKCZ; HDAC4; TGM2; Signaling MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5; CREB1; PRKCI; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1; GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11; MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1; CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CLTC; SGK; HDAC6; CASP3 Apoptosis PRKCE; ROCK1; BID; IRAK1; Signaling PRKAA2; EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; .IKBKB; CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2LI; CAPN1; MAPK3; CASPS; KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAFI; IKBKG; RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKBI; PAK3; LMNA; CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 B Cell RACI; PTEN; LYN; Receptor ELK1; MAPK1; RAC2; PTPN11 ; Signaling AKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3; MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9; EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN; GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Leukocyte ACTN4; CD44; PRKCE; Extravasation ITGAM; ROCK1; CXCR4; CYBA; Signaling RAC1; RAP1A.; PRKCZ; ROCK2; RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8; PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A.; BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1 CTNNB1.; CLDN1 CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9 Integrin ACTN4; ITGAM; ROCK1; Signaling ITGA5; RAC1; PTEN; RAP1A; TLN1; ARHGEF7; MAPKI; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3; MAPK8; CAV1; CAPN1; ABLI; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7; PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3RI; TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3 Acute IRAK1; SOD2; MYD88; Phase TRAF6; ELK1; Response MAPK1; PTPN11; Signaling AKT2; .IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8; RIPK1; MAPK3; 1L6ST; KRAS; MAPK13; IL6R; RELA; SOCSI; MAPK9; FTL; NR3C1; TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3; IL1Rl.; IL6 PTEN ITGAM; ITGA5; RAC1; Signaling PTEN; PRKCZ; BCL2L11; MAPKI; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2; PIK3CB; BCL2L1; MAPK3; ITGA1; .KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1; IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1; MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1; CASP3; RPS6KB1 p53
Signaling PTEN; EP300; BBC3; PCAF; FASN; BRCAT; GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3; MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1.; CHEK2; TNFRSF10B; TP73; RBI; HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1 ; PIK3R1; RRM213; APAFI; CTNNB1; SIRT1; CCND1 ; .PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2; GSK3B; BAX; AKT3 Aryl HSPB1; EP300; FASN; Hydrocarbon TGM2; RXRA; MAPK1; NQO1; Receptor NCOR2; SP1; ARNT; Signaling CDKN1B; FOS; CHEK1; SMARCA4; NFKB2; MAPK8; ALDH1A1.; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73; GSTP1; RB1; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2; APAF1; NFKB1 ; CCND1 ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; 1L6; CYP1B1; HSP90AA1 Xenobiotic PRKCE; EP300; PRKCZ; Metabolism RXRA; MAPK1; NQO1; Signaling NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB; PPP2R1A; PIK3C3; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13; PRKCD; GSTP1;MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; P1K3C2A; PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1; NFKBI; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AAI SAPK/JNK PRKCE; IRAK1; PRKAA2; Signaling EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2; PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1; IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MARK9; CDK2; PIM1; PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3; CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK PPAr/RXR PRKAA2; EP300; INS; Signaling SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB; NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS; RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR; RAFT; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1; ADIPOQ NF-KB IRAK1; EIF2AK2; EP300; Signaling INS; MYD88; PRKCZ; TRAF6; TBKI; AKT2; EGFR IKBKB; PIK3CA; BTRC; NFKB2; MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A; TRAF2; TLR4; PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1; PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1 Neuregulin ERBB4; PRKCE; ITGAM; Signaling ITGA5; PTEN; PRKCZ; ELK1; MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3; ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1.; MAP2K2; ADAM 17; AKT1; PIK3R1; PDPKI ; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC; NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt & Beta CD44; EP300; LRP6; catenin DVL3; CSNK1E; GJA1; SMO; Signaling AKT2; PIN1; CDH1; BTRC; GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2; ILK; LEF1; SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1;TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2 Insulin PTEN; INS; EIF4E; Receptor PTPN1; PRKCZ; MAPK1; TSC1; Signaling PTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3; TSC2; KRAS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B; AKT3; FOXO1; SGK; RPS6KB1 1L-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1; MAPKi; PTPN11; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1; CEBPB; JUN; IL1RI; SRF; IL6 Hepatic PRKCE; IRAK1; INS; MYD88; Cholestasis PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8; PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKI31; ESR1; SREBF1; FGFR4; JUN; ILIRI; PRKCA.; 1L6 IGF-1 IGF1; PRKCZ; ELK1; Signaling MAPK1; PTPN11; NEDD4; AKT2; PIK3CA; PRKC1; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R; IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR6I; AKT3; FOXO1; SRF; CTGF; RPS6KB1 NRF2-mediated PRKCE; EP300; SOD2; Oxidative PRKCZ; MAPKI; SQSTM1; Stress NQO1; PIK3CA; PRKCI; FOS; Response PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A; MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; P1K3R1; MAP2K1; PPIB; JUN; KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic EDN1; IGF1; KDR; FLT1; Fibrosis/ SMAD2; FGFR1; MET; PGF; Hepatic SMAD3; EGFR; FAS; CSF1; Stellate NFKB2; BCL2; MYH9; Cell IGHF1R; IL6R; RELA; TLR4; Activation PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1; CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 PPAR EP300; INS; TRAF6; PPARA; Signaling RXRA; MAPK1; IKBKG; NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3; NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF; INSR; RAF1; IKBKG; RELB; MAP3K7- CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1; NFKB1; JUN; IL1R1; HSP90AA1 Fc Epsilon PRKCE; RAC1; PRKCZ; RI Signaling LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; P1K3CB; PIK3C3; MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK; MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; P1K3R1; PDPK1; MAP2K1; AKT3; VAV3; PRKCA G-Protein PRKCE; .RAP1A ; RGS16; Coupled MAPK1; GNAS; AKT2; IKBKB; Receptor PIK3CA; CREB1; GNAQ; Signaling NFKB2; CAMK2A; PIK3CB; PIK3C3: MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1; PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCA Inositol PRKCE; IRAK1; PRKAA2; Phosphate EIF2AK2; PTEN; GRK6; Metabolism MAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1; MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK PDGF EIF2AK2; ELK1; ABL2.; Signaling MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3; KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA; STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGF ACTN4; ROCK1; KDR; Signaling FLT1.; ROCK2; MAPK1; PGF;
AKT2; PIK3CA; ARNT; PTK2; BCL2; PIK3CB; P1K3C3;BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1.; SFN; VEGFA; AKT3; FOXO1; PRKCA Natural PRKCE; RAC1; Killer Cell PRKCZ; MAPK1; Signaling RAC2; PTPN11; KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; PRKD1; MAPK3; KRAS; PRKCD; PTPN6; P1K3C2A; LCK; RAF1; FYN; MAP2K2; PAK4; AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: HDAC4; SMAD3; G1/S SUV39H1; HDAC5; CDKN1B; BTRC; Checkpoint ATR; ABL1; E2F1; Regulation HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1; HDAC6 T Cell RAC1; ELK1; MAPK1; Receptor IKBKB; CBL; Signaling PIK3CA; FOS; NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA; PIK3C2A; BTK; LCK.; RAF1; IKBKG; RELB; FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK; BCL10; JUN; VAV3 Death CRADD; HSPB1; BID; Receptor BIRC4; TBK1; IKBKB; Signaling FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX; TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKBI; CASP2; BIRC2; CASP3; BIRC3 FGF RAC1; FGFR1; MET; Signaling MAPKAPK2; MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3; MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1; FGFR4; CRKL; ATF4; AKT3; PRKCA; HCF GM-CSF LYN; ELK1; MAPK1; Signaling PTPN11; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1; MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Amyotrophic BID; IGF1; RAC1; Lateral BIRC4; PGF; Sclerosis CAPNS1; CAPN2; Signaling PIK3CA; BCL2; PIK3CB; PIK30; BCL2L1.; CAPNI; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1; APAF1 ; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/ PTPNI; MAPK1; Stat PTPN11; AKT2; PIK3CA; Signaling STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS; SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; FRAPI; AKT3; STAT1 Nicotinate PRKCE; IRAK1; and PRKAA2; EIF2AK2; Nicotinamide GRK6; MAPK1; Metabolism PLK1; AKT2; CDK8; MAPK8; MAPK3; PRKCD; PRKAA1; PBEF1; MAPK9; CDK2; PIM1; DYRK1A; MAP2K2;MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine CXCR4; ROCK2; MAPK1; Signaling PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12.; MAPK8; MAPK3; KRAS; MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1; JUN; CCL2; PRKCA 1L-2 ELK1; MAPK1; Signaling PTPN11; AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; SOCSI; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3 Synaptic PRKCE; IGF1; PRKCZ; Long PRDX6; LYN; MAPK1; Term GNAS; Depression PRKCI; GNAQ; PPP2R1A; IGFIR; PRKDI; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCA Estrogen TAF4B; EP300; CARM1; Receptor PCAF; MAPK1; NCOR2; Signaling SMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 Protein TRAF6; SMURF1; BIRC4; Ubiquitination BRCA1; UCHLI; NEDD4; Pathway CBL; UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1; VHL; HSP90AA1; BIRC3 IL-10 Signaling TRAF6; CCR1; ELK1; IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6 VDR/ PRKCE; EP300; RXR PRKCZ; RXRA; Activation GADD45A; HES1; NCOR2; SP1; PRKCI; CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1; LRP5; CEBPB; FOXO1; PRKCA TGF- EP300; SMAD2; beta SMURF1; MAPK1; Signaling SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2.; SERPINE1; RAF1; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5 Toll- IRAKI; EIF2AK2; like MYD88; TRAF6; Receptor PPARA; ELK1; Signaling IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN p38 HSPB1; IRAK1; TRAF6; MAPK Signaling MAPKAPK2; ELK1; FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK 14; TNF; MAP3K7; TGFBR1; MYC; ATF4; 1L1R1 ; SRF; STAT1 Neurotrophin/ NTRK2; MAPK1; PTPN11 ; TRK PIK3CA; CREB1; FOS; Signaling PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42; JUN; ATF4 FXR/ INS; PPARA; FASN; RXR RXRA; AKT2; Activation SDC1; MAPK8; APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; FOXO1 Synaptic PRKCE; RAP1A; Long EP300; PRKCZ; Term MAPK1; CREB1; Potentiation PRKCI; GNAQ; CAMK2A; PRKD1; MAPK3; KRAS; PRKCD; PPP1CC; RAFI; CREBBP; MAP2K2; MAP2K1; ATF4; PRKCA Calcium RAP1A; EP300; Signaling HDAC4; MAPK1; HDAC5; CREB1; CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4;
HDAC6 EGF ELK1; MAPK1; EGFR; Signaling PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1 Hypoxia EDN1; PTEN; EP300; Signaling NQO1; UBE2I; in the CREB1; ARNT; Cardiovascular HIF1A; SLC2A4; System NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN; ATF4; VHL; HSP90AA1 LPS/ IRAK1; MYD88; IL-1 TRAF6; PPARA; Mediated. RXRA; ABCA1; Inhibition MAPK8; ALDH1A1; of RXR Function GSTP1; MAPK9; ABCB1; TRAF2; TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN; IL1R1 LXR/ FASN; RXRA; RXR NCOR2; ABCA1.; Activation NFKB2; IRF3; RELA; NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1; IL1R1; CCL2; IL6; MMP9 Amyloid PRKCE; CSNK1E; Processing MAPK1; CAPNS1; AKT2; CAPN2; CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1; GSK3B; AKT3; APP 1L-4 AKT2; PIK3CA; PIK3CB; Signaling PIK3C3; IRS1; KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1; AKT3; RPS6KB1 Cell Cycle: EP300; PCAF; G2/M BRCA1; GADD45A; DNA PLK1; BTRC; Damage CHEK1; ATR; CHEK2; Checkpoint YWHAZ; TP53; CDKN1A; Regulation PRKDC; ATM; SFN; CDKN2A Nitric KDR; FLT1; PGF; Oxide AKT2; PIK3CA; Signaling PIK3CB; PIK3C3; in CAV1; PRKCD; NOS3; PIK3C2A; the AKT1; PIK3R1; Cardiovascular System VEGFA.; AKT3; HSP90AA1 Purine NME2; SMARCA4; Metabolism MYH9; RRM2; ADAR.; EIF2AK4 PKM2; ENTPD1; RAD51; RRM2B; TJP2; .RAD51C; NT5E; POLD1; NME1 cAMP- RAP1A; MAPK1; GNAS; mediated CREB1; CAMK2A; MAPK3; Signaling SRC; RAH; MAP2K2; STAT3; MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8; Dysfunction CASP8;MAPK10; MAPK9 CASP9; PARK7; PSEN1; PARK2; APP; CASP3 Notch HES1; JAG1; NUMB; Signaling NOTCH4; ADAM17; NOTCH2; PSENI; NOTCH3; NOTCHI; DLL4 Endoplasmic HSPA5; MAPK8; Reticulum XBP1; TRAF2; Stress ATF6; CASP9; ATF4; Pathway ElF2AK3; CASP3 Pyrimidine NME2; AICDA; RRM2; Metabolism EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NMEI Parkinson`s UCHL1; MAPK8; MAPK13; Signaling MAPK14; CASP9; PARK7; PARK2; CASP3 Cardiac & GNAS; GNAQ; PPP2R1A; Beta GNB2L1; PPP2CA; Adrenergic PPP1CC; Signaling PPP2R5C Glycolysis/ HK2; GCK; GPI; Gluconeogenesis ALDH1A1; PKM2; LDHA; HK1 Interferon IRF1; SOCS1; JAK1; Signaling JAK2; IFITM1; STAT1; IFIT3 Sonic ARRB2; SMO; GLI2; Hedgehog DYRK1A; GLI1; Signaling GSK3B; DYRK1B Glycero- PLD1; GRN; phospholipid. GPAM; YWHAZ; Metabolism SPHK1; SPHK2 Phospholipid PRDX6; PLD1; GRN; Degradation YWHAZ; SPHK1; SPHK2 Tryptophan SIAH2; PRMT5; NEDD4; Metabolism ALDH1A1; CYP1B1; SIAH1 Lysine SUV39H1; EHMT2; Degradation NSD1; SETD7; PPP2R5C Nucleotide ERCC5; ERCC4; Excision XPA; XPC; ERCC1 Repair Pathway Starch and UCHL1; HK2; Sucrose GCK; GPI; HK1 Metabolism Aminosugars NQO1; HK2; Metabolism GCK; HK1 Arachidonic Acid PRDX6; Metabolism GRN; YWHAZ; CYP1B1 Circadian CSNK1E; CREB1; Rhythm ATF4; NR1D1 Signaling Coagulation BDKRB1; F2R; System SERPINE1; F3 Dopamine PPP2R1A; PPP2CA; Receptor PPP1CC; PPP2R5C Signaling Glutathione IDH2; GSTP1; Metabolism ANPEP; IDH1 Glycerolipid ALDH1A1; GPAM; Metabolism SPHK1; SPHK2 Linoleic PRDX6; GRN; Acid YWHAZ; CYP1B1 Metabolism Methionine DNMT1; DNMT3B; Metabolism. AHCY; DNMT3A Pyruvate GLO1; ALDH1A1; Metabolism PKM2; LDHA Arginine ALDH1A1; and Proline NOS3; NOS2A Metabolism Eicosanoid PRDX6; GRN; Signaling YWHAZ Fructose HK2; GCK; and Mannose HK1 Metabolism Galactose HK2; GCK; Metabolism HK1 Stilbene, PRDX6; PRDX1; Cournarine and TYR Lignin Biosynthesis Antigen Presentation CALR; B2M Pathway Biosynthesis of Steroids NQO1; DHCR7 Butanoate Metabolism ALDH1A1; NLGN1 Citrate Cycle IDH2; IDH1 Fatty Acid Metabolism ALDH1A1; CYP1B1 Glycerophospholipid PRDX6; CHKA Metabolism Histidine Metabolism PRMT5; ALDH1A1 Inositol Metabolism ERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics by Cytochrome p450 Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6; PRDX1 Metabolism Propanoate Metabolism ALDH1A1; LDHA Selenoamino Acid PRMT5; AHCY Metabolism Sphingolipid Metabolism SPHK1; SPHK2 Aminophosphonate PRMT5 Metabolism Androgen and Estrogen PRMT5 Metabolism Ascorbate and Aldarate ALDH1A1 Metabolism Bile Acid Biosynthesis ALDH1A1 Cysteine Metabolism LDHA Fatty Acid Biosynthesis FASN Glutamate Receptor GNB2L1 Signaling NRF2-mediated PRDX1 Oxidative Stress Response Pentose Phosphate GPI Pathway Pentose and Glucuronate UCHL1 interconversions Retinol Metabolism ALDH1A1 Riboflavin Metabolism TYR Tyrosine Metabolism PRMT5, TYR Ubiquinone Biosynthesis PRMT5 Valine, Leucine and ALDH1A1 Isoleucine Degradation Glycine, Serine and CHKA Threonine Metabolism Lysine Degradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain TRPM7; TRPC5; TRPC6; TRPC1; Cnrl; cnr2; Grk2; Trp1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5; Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial AIF; CytC; SMAC (Diablo); Function Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd); Neurology Noggin (Nog); WNT (Wnt2; Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b; Wnt8b; Wnt9a; Wnt9b; Wnt10a; Witt10b; Wnt16); beta-catenin; Dkk-1; Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1; unc-86 (Pou4fl or Brn3a); Numb; Reln
[0126] Embodiments of the invention also relate to methods and compositions related to knocking out genes, amplifying genes and repairing particular mutations associated with DNA repeat instability and neurological disorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities and Neurological Diseases, Second Edition, Academic Press, Oct. 13, 2011--Medical). Specific aspects of tandem repeat sequences have been found to be responsible for more than twenty human diseases (New insights into repeat instability: role of RNA*DNA hybrids. Melvor E I, Polak U. Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). The CRISPR-Cas system may be harnessed to correct these defects of genomic instability.
[0127] A further aspect of the invention relates to utilizing the CRISPR-Cas system for correcting defects in the EMP2A and EMP2B genes that have been identified to be associated with Lafora disease. Lafora disease is an autosomal recessive condition which is characterized by progressive myoclonus epilepsy which may start as epileptic seizures in adolescence. A few cases of the disease may be caused by mutations in genes yet to be identified. The disease causes seizures, muscle spasms, difficulty walking, dementia, and eventually death. There is currently no therapy that has proven effective against disease progression. Other genetic abnormalities associated with epilepsy may also be targeted by the CRISPR-Cas system and the underlying genetics is further described in Genetics of Epilepsy and Genetic Epilepsies, edited by Giuliano Avanzini, Jeffrey L. Noebels, Mariani Foundation Paediatric Neurology: 20; 2009).
[0128] In yet another aspect of the invention, the CRISPR-Cas system may be Cl used to correct ocular defects that arise from several genetic mutations further described in Genetic Diseases of the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford University Press, 2012.
[0129] Several further aspects of the invention relate to correcting defects associated with a wide range of genetic diseases which are further described on the website of the National Institutes of Health under the topic subsection Genetic Disorders. The genetic brain diseases may include but are not limited to Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Aicardi Syndrome, Alpers' Disease, Alzheimer's Disease, Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration, Fabry's Disease, Gerstmann-Straussler-Scheinker Disease, Huntington's Disease and other Triplet Repeat Disorders, Leigh's Disease, Lesch-Nyhan Syndrome, Menkes Disease, Mitochondrial Myopathies and NINDS Colpocephaly. These diseases are further described on the website of the National Institutes of Health under the subsection Genetic Brain Disorders.
[0130] In some embodiments, the condition may be neoplasia. In some embodiments, where the condition is neoplasia, the genes to be targeted are any of those listed in Table A (in this case PTEN asn so forth). In some embodiments, the condition may be Age-related Macular Degeneration. In some embodiments, the condition may be a Schizophrenic Disorder. In some embodiments, the condition may be a Trinucleotide Repeat Disorder. In some embodiments, the condition may be Fragile X Syndrome. In some embodiments, the condition may be a Secretase Related Disorder. In some embodiments, the condition may be a Prion--related disorder. In some embodiments, the condition may be ALS. In some embodiments, the condition may be a drug addiction. In some embodiments, the condition may be Autism. In some embodiments, the condition may be Alzheimer's Disease. In some embodiments, the condition may be inflammation. In some embodiments, the condition may be Parkinson's Disease.
[0131] Examples of proteins associated with Parkinson's disease include but are not limited to α-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1, Synphilin-1, and NURR1.
[0132] Examples of addiction-related proteins may include ABAT for example.
[0133] Examples of inflammation-related proteins may include the monocyte chemoattractant protein-1 (MCP1) encoded by the Ccr2 gene, the C-C chemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, the IgG receptor IIB (FCGR2b, also termed CD32) encoded by the Fcgr2b gene, or the Fc epsilon R1g (FCER1g) protein encoded by the Fcer1g gene, for example.
[0134] Examples of cardiovascular diseases associated proteins may include IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53). PTGIS (prostaglandin I2 (prostacyclin) synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-binding cassette, sub-family G (WHITE), member 8), or CTSK (cathepsin K), for example.
[0135] Examples of Alzheimer's disease associated proteins may include the very low density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, the ubiquitin-like modifier activating enzyme 1 (UBA1) encoded by the UBA1 gene, or the NEDD8-activating enzyme E1 catalytic subunit protein (UBE1C) encoded by the UBA3 gene, for example.
[0136] Examples of proteins associated with Autism Spectrum Disorder may include the benzodiazapine receptor (peripheral) associated protein 1 (BZRAP1) encoded by the BZRAP1 gene, the AF4/FMR2 family member 2 protein (AFF2) encoded by the AFF2 gene (also termed MFR2), the fragile X mental retardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene, or the fragile X mental retardation autosomal homolog 2 protein (FXR2) encoded by the FXR2 gene, for example.
[0137] Examples of proteins associated with Macular Degeneration may include the ATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4) encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded by the APOE gene, or the chemokine (C-C motif) Ligand 2 protein (CCL2) encoded by the CCL2 gene, for example.
[0138] Examples of proteins associated with Schizophrenia may include NRG1, ErbB4, CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, and combinations thereof.
[0139] Examples of proteins involved in tumor suppression may include ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch2, Notch 3, or Notch 4, for example.
[0140] Examples of proteins associated with a secretase disorder may include PSENEN (presenilin enhancer 2 homolog (C. elegans)), CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid beta (A4) precursor protein), APH1B (anterior pharynx defective 1 homolog B (C. elegans)), PSEN2 (presenilin 2 (Alzheimer disease 4)), or BACE1 (beta-site APP-cleaving enzyme 1), for example.
[0141] Examples of proteins associated with Amyotrophic Lateral Sclerosis may include SOD1 (superoxide dismutase 1). ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof.
[0142] Examples of proteins associated with prion diseases may include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof.
[0143] Examples of proteins related to neurodegenerative conditions in prion disorders may include A2M (Alpha-2-Macroglobulin), AATF (Apoptosis antagonizing transcription factor), ACPP (Acid phosphatase prostate), ACTA2 (Actin alpha 2 smooth muscle aorta), ADAM22 (ADAM metallopeptidase domain), ADORA3 (Adenosine A3 receptor), or ADRA1D (Alpha-1D adrenergic receptor for Alpha-1D adrenoreceptor), for example.
[0144] Examples of proteins associated with Immunodeficiency may include A2M [alpha-2-macroglobulin]; AANAT [arylalkylamine N-acetyltransferase]; ABCA1 [ATP-binding cassette, sub-family A (ABC1), member 1]; ABCA2 [ATP-binding cassette, sub-family A (ABC1), member 2]; or ABCA3 [ATP-binding cassette, sub-family A (ABC1), member 3]; for example.
[0145] Examples of proteins associated with Trinucleotide Repeat Disorders include AR (androgen receptor), FMR1 (fragile X mental retardation 1), HTT (huntingtin), or DMPK (dystrophia myotonica-protein kinase), FXN (frataxin), ATXN2 (ataxin 2), for example.
[0146] Examples of proteins associated with Neurotransmission Disorders include SST (somatostatin), NOS1 (nitric oxide synthase 1 (neuronal)), ADRA2A (adrenergic, alpha-2A-, receptor), ADRA2C (adrenergic, alpha-2C-, receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-hydroxytryptamine (serotonin) receptor 2C), for example.
[0147] Examples of neurodevelopmental-associated sequences include A2BP1 [ataxin 2-binding protein 1], AADAT [aminoadipate aminotransferase], AANAT [arylalkylamine N-acetyltransferase], ABAT [4-aminobutyrate aminotransferase], ABCA1 [ATP-binding cassette, sub-family A (ABC1), member 1], or ABCA13 [ATP-binding cassette, sub-family A (ABC1), member 13], for example.
[0148] Further examples of preferred conditions treatable with the present system include may be selected from: Aicardi-Goutieres Syndrome; Alexander Disease; Allan-Herndon-Dudley Syndrome; POLG-Related Disorders; Alpha-Mannosidosis (Type II and III); Alstr6m Syndrome; Angelman; Syndrome; Ataxia-Telangiectasia; Neuronal Ceroid-Lipofuscinoses; Beta-Thalassemia; Bilateral Optic Atrophy and (Infantile) Optic Atrophy Type 1; Retinoblastoma (bilateral); Canavan Disease; Cerebrooculofacioskeletal Syndrome 1 [COFS 1]; Cerebrotendinous Xanthomatosis; Cornelia de Lange Syndrome; MAPT-Related Disorders; Genetic Prion Diseases; Dravet Syndrome; Early-Onset Familial Alzheimer Disease; Friedreich Ataxia [FRDA]; Fryns Syndrome; Fucosidosis; Fukuyama Congenital Muscular Dystrophy; Galactosialidosis; Gaucher Disease; Organic Acidemias; Hemophagocytic Lymphohistiocytosis; Hutchinson-Gilford Progeria Syndrome; Mucolipidosis 11; Infantile Free Sialic Acid Storage Disease; PLA2G6-Associated Neurodegeneration; Jervell and Lange-Nielsen Syndrome; Junctional Epidermolysis Bullosa; Huntington Disease; Krabbe Disease (Infantile); Mitochondrial DNA-Associated Leigh Syndrome and NARP; Lesch-Nyhan Syndrome; LIS1-Associated Lissencephaly; Lowe Syndrome; Maple Syrup Urine Disease; MECP2 Duplication Syndrome; ATP7A-Related Copper Transport Disorders; LAMA2-Related Muscular Dystrophy; Arylsulfatase A Deficiency; Mucopolysaccharidosis Types I, II or III; Peroxisome Biogenesis Disorders, Zellweger Syndrome Spectrum; Neurodegeneration with Brain Iron Accumulation Disorders; Acid Sphingomyelinase Deficiency; Niemann-Pick Disease Type C; Glycine Encephalopathy; ARX-Related Disorders; Urea Cycle Disorders; COL1A1/2-Related Osteogenesis Imperfecta; Mitochondrial DNA Deletion Syndromes; PLP1-Related Disorders; Perry Syndrome; Phelan-McDermid Syndrome; Glycogen Storage Disease Type II (Pompe Disease) (Infantile); MAPT-Related Disorders; MECP2-Related Disorders; Rhizomelic Chondrodysplasia Punctata Type 1; Roberts Syndrome; Sandhoff Disease; Schindler Disease--Type 1; Adenosine Deaminase Deficiency; Smith-Lemli-Opitz Syndrome; Spinal Muscular Atrophy; Infantile-Onset Spinocerebellar Ataxia; Hexosaminidase A Deficiency; Thanatophoric Dysplasia Type 1; Collagen Type VI-Related Disorders; Usher Syndrome Type I; Congenital Muscular Dystrophy; Wolf-Hirschhorn Syndrome; Lysosomal Acid Lipase Deficiency; and Xeroderma Pigmentosum.
[0149] Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. The immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes. Reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity (Tangri S, et al. ("Rationally engineered therapeutic proteins with reduced immunogenicity" J Immunol. 2005 Mar. 15; 174(6):3187-96.) In the present invention, the immunogenicity of the CRISPR enzyme in particular may be reduced following the approach first set out in Tangri et al with respect to erythropoietin and subsequently developed. Accordingly, directed evolution or rational design may be used to reduce the immunogenicity of the CRISPR enzyme (for instance a Cas9) in the host species (human or other species).
[0150] In plants, pathogens are often host-specific. For example, Fusarium oxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato, and F. oxysporum f dianthii Puccinia graminis f. sp. tritici attacks only wheat. Plants have existing and induced defenses to resist most pathogens. Mutations and recombination events across plant generations lead to genetic variability that gives rise to susceptibility, especially as pathogens reproduce with more frequency than plants. In plants there can be non-host resistance, e.g., the host and pathogen are incompatible. There can also be Horizontal Resistance, e.g., partial resistance against all races of a pathogen, typically controlled by many genes and Vertical Resistance, e.g., complete resistance to some races of a pathogen but not to other races, typically controlled by a few genes. In a Gene-for-Gene level, plants and pathogens evolve together, and the genetic changes in one balance changes in other. Accordingly, using Natural Variability, breeders combine most useful genes for Yield, Quality, Uniformity, Hardiness, Resistance. The sources of resistance genes include native or foreign Varieties, Heirloom Varieties, Wild Plant Relatives, and Induced Mutations, e.g., treating plant material with mutagenic agents. Using the present invention, plant breeders are provided with a new tool to induce mutations. Accordingly, one skilled in the art can analyze the genome of sources of resistance genes, and in Varieties having desired characteristics or traits employ the present invention to induce the rise of resistance genes, with more precision than previous mutagenic agents and hence accelerate and improve plant breeding programs.
[0151] As will be apparent, it is envisaged that the present system can be used to target any polynucleotide sequence of interest. Some examples of conditions or diseases that might be usefully treated using the present system are included in the Tables above and examples of genes currently associated with those conditions are also provided there. However, the genes exemplified are not exhaustive.
EXAMPLES
[0152] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
Example 1
CRISPR Complex Activity in the Nucleus of a Eukaryotic Cell
[0153] An example type II CRISPR system is the type 11 CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30 bp each). In this system, targeted DNA double-strand break (DSB) is generated in four sequential steps (FIG. 2A). First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the direct repeats of pre-crRNA, which is then processed into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer (FIG. 2A). This example describes an example process for adapting this RNA-programmable nuclease system to direct CRISPR complex activity in the nuclei of eukaryotic cells.
[0154] To improve expression of CRISPR components in mammalian cells, two genes from the SF370 locus 1 of Streptococcus pyogenes (S. pyogenes) were codon-optimized, Cas9 (SpCas9) and RNase III (SpRNase III). To facilitate nuclear localization, a nuclear localization signal (NLS) was included at the amino (N)- or carboxyl (C)-termini of both SpCas9 and SpRNase III (FIG. 2B). To facilitate visualization of protein expression, a fluorescent protein marker was also included at the N- or C-termini of both proteins (FIG. 2B). A version of SpCas9 with an NLS attached to both N- and C-termini (2xNLS-SpCas9) was also generated. Constructs containing NLS-fused SpCas9 and SpRNase III were transfected into 293FT human embryonic kidney (HEK) cells, and the relative positioning of the NLS to SpCas9 and SpRNase III was found to affect their nuclear localization efficiency. Whereas the C-terminal NLS was sufficient to target SpRNase III to the nucleus, attachment of a single copy of these particular NLS's to either the N- or C-terminus of SpCas9 was unable to achieve adequate nuclear localization in this system. In this example, the C-terminal NLS was that of nucleoplasmin (KRPAATKKAGQAKKKK), and the C-terminal NLS was that of the SV40 large T-antigen (PKKKRKV). Of the versions of SpCas9 tested, only 2xNLS-SpCas9 exhibited nuclear localization (FIG. 2B).
[0155] The tracrRNA from the CRISPR locus of S. pyogenes SF370 has two transcriptional start sites, giving rise to two transcripts of 89-nucleotides (nt) and 171nt that are subsequently processed into identical 75nt mature tracrRNAs. The shorter 89nt tracrRNA was selected for expression in mammalian cells (expression constructs illustrated in FIG. 6, with functionality as determined by results of Surveryor assay shown in FIG. 6B). Transcription start sites are marked as +1, and transcription terminator and the sequence probed by northern blot are also indicated. Expression of processed tracrRNA was also confirmed by Northern blot. FIG. 7C shows results of a Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying long or short tracrRNA, as well as SpCas9 and DR-EMX1(1)-DR. Left and right panels are from 293FT cells transfected without or with SpRNase III, respectively. U6 indicate loading control blotted with a probe targeting human U6 snRNA. Transfection of the short tracrRNA expression construct led to abundant levels of the processed form of tracrRNA (˜75 bp). Very low amounts of long tracrRNA are detected on the Northern blot.
[0156] To promote precise transcriptional initiation, the RNA polymerase III-based U6 promoter was selected to drive the expression of tracrRNA (FIG. 2C). Similarly, a U6 promoter-based construct was developed to express a pre-crRNA array consisting of a single spacer flanked by two direct repeats (DRs, also encompassed by the term "tracr-mate sequences"; FIG. 2C). The initial spacer was designed to target a 33-base-pair (bp) target site (30-bp protospacer plus a 3-bp CRISPR motif (PAM) sequence satisfying the NGG recognition motif of Cas9) in the human EALX1 locus (FIG. 2C), a key gene in the development of the cerebral cortex.
[0157] To test whether heterologous expression of the CRISPR system (SpCas9, SpRNase III, tracrRNA, and pre-crRNA) in mammalian cells can achieve targeted cleavage of mammalian chromosomes, HEK 293FT cells were transfected with combinations of CRISPR components. Since DSBs in mammalian nuclei are partially repaired by the non-homologous end joining (NHEJ) pathway, which leads to the formation of indels, the Surveyor assay was used to detect potential cleavage activity at the target EMX1 locus (see e.g. Guschin et al., 2010, Methods Mol Biol 649: 247). Co-transfection of all four CRISPR components was able to induce up to 5.0% cleavage in the protospacer (see FIG. 2D). Co-transfection of all CRISPR components minus SpRNase III also induced up to 4.7% indel in the protospacer, suggesting that there may be endogenous mammalian RNases that are capable of assisting with crRNA maturation, such as for example the related Dicer and Drosha enzymes. Removing any of the remaining three components abolished the genome cleavage activity of the CRISPR system (FIG. 2D). Sanger sequencing of amplicons containing the target locus verified the cleavage activity: in 43 sequenced clones, 5 mutated alleles (11.6%) were found. Similar experiments using a variety of guide sequences produced indel percentages as high as 29% (see FIGS. 4-8, 10 and 11). These results define a three-component system for efficient CRISPR-mediated genome modification in mammalian cells.
[0158] To optimize the cleavage efficiency, Applicants also tested whether different isoforms of tracrRNA affected the cleavage efficiency and found that, in this example system, only the short (89-bp) transcript form was able to mediate cleavage of the human EM-AV genomic locus. FIG. 9 provides an additional Northern blot analysis of crRNA processing in mammalian cells. FIG. 9A illustrates a schematic showing the expression vector for a single spacer flanked by two direct repeats (DR-EMX1(1)-DR). The 30 bp spacer targeting the human EMX1 locus protospacer 1 and the direct repeat sequences are shown in the sequence beneath FIG. 9A. The line indicates the region whose reverse-complement sequence was used to generate Northern blot probes for EMX1(1) crRNA detection. FIG. 9B shows a Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying DR-EMX1(1)-DR. Left and right panels are from 293FT cells transfected without or with SpRNase III respectively. DR-EMX1(1)-DR was processed into mature crRNAs only in the presence of SpCas9 and short tracrRNA and was not dependent on the presence of SpRNase III. The mature crRNA detected from transfected 293FT total RNA is ˜33 bp and is shorter than the 39-42 bp mature crRNA from S. pyogenes. These results demonstrate that a CRISPR system can be transplanted into eukaryotic cells and reprogrammed to facilitate cleavage of endogenous mammalian target polynucleotides.
[0159] FIG. 2 illustrates the bacterial CRISPR system described in this example. FIG. 2A illustrates a schematic showing the CRISPR locus 1 from Streptococcus pyogenes SF370 and a proposed mechanism of CRISPR-mediated DNA cleavage by this system. Mature crRNA processed from the direct repeat-spacer array directs Cas9 to genomic targets consisting of complimentary protospacers and a protospacer-adjacent motif (PAM). Upon target-spacer base pairing, Cas9 mediates a double-strand break in the target DNA. FIG. 2B illustrates engineering of S. pyogenes Cas9 (SpCas9) and RNase III (SpRNase III) with nuclear localization signals (NLSs) to enable import into the mammalian nucleus. FIG. 2C illustrates mammalian expression of SpCas9 and SpRNase III driven by the constitutive EF1a promoter and tracrRNA and pre-crRNA array (DR-Spacer-DR) driven by the RNA Pol3 promoter U6 to promote precise transcription initiation and termination. A protospacer from the human EMX1 locus with a satisfactory PAM sequence is used as the spacer in the pre-crRNA array. FIG. 2D illustrates surveyor nuclease assay for SpCas9-mediated minor insertions and deletions. SpCas9 was expressed with and without SpRNase III, tracrRNA, and a pre-crRNA array carrying the EMX1-target spacer. FIG. 2E illustrates a schematic representation of base pairing between target locus and EMX1-targeting crRNA, as well as an example chromatogram showing a micro deletion adjacent to the SpCas9 cleavage site. FIG. 2F illustrates mutated alleles identified from sequencing analysis of 43 clonal amplicons showing a variety of micro insertions and deletions. Dashes indicate deleted bases, and non-aligned or mismatched bases indicate insertions or mutations. Scale bar=10 μm.
[0160] To further simplify the three-component system, a chimeric crRNA-tracrRNA hybrid design was adapted, where a mature crRNA (comprising a guide sequence) is fused to a partial tracrRNA via a stem-loop to mimic the natural crRNA:tracrRNA duplex (FIG. 3A).
[0161] Guide sequences can be inserted between BbsI sites using annealed oligonucleotides. Protospacers on the sense and anti-sense strands are indicated above and below the DNA sequences, respectively. A modification rate of 6.3% and 0.75% was achieved for the human PVALB and mouse Th loci respectively, demonstrating the broad applicability of the CRISPR system in modifying different loci across multiple organisms While cleavage was only detected with one out of three spacers for each locus using the chimeric constructs, all target sequences were cleaved with efficiency of indel production reaching 27% when using the co-expressed pre-crRNA arrangement (FIGS. 4 and 5).
[0162] FIG. 5 provides a further illustration that SpCas9 can be reprogrammed to target multiple genomic loci in mammalian cells. FIG. 5A provides a schematic of the human EMX1 locus showing the location of five protospacers, indicated by the underlined sequences. FIG. 5B provides a schematic of the pre-crRNA/trcrRNA complex showing hybridization between the direct repeat region of the pre-crRNA and tracrRNA (top), and a schematic of a chimeric RNA design comprising a 20 bp guide sequence, and tracr mate and tracr sequences consisting of partial direct repeat and tracrRNA sequences hybridized in a hairpin structure (bottom). Results of a Surveyor assay comparing the efficacy of Cas9-mediated cleavage at five protospacers in the human EMX1 locus is illustrated in FIG. 5C. Each protospacer is targeted using either processed pre-crRNA/tracrRNA complex (crRNA) or chimeric RNA (chiRNA).
[0163] Since the secondary structure of RNA can be crucial for intermolecular interactions, a structure prediction algorithm based on minimum free energy and Boltzmann-weighted structure ensemble was used to compare the putative secondary structure of all guide sequences used in our genome targeting experiment (FIG. 3B) (see e.g. Gruber et al., 2008, Nucleic Acids Research, 36: W70). Analysis revealed that in most cases, the effective guide sequences in the chimeric crRNA context were substantially free of secondary structure motifs, whereas the ineffective guide sequences were more likely to form internal secondary structures that could prevent base pairing with the target protospacer DNA. It is thus possible that variability in the spacer secondary structure might impact the efficiency of CRISPR-mediated interference when using a chimeric crRNA.
[0164] FIG. 3 illustrates example expression vectors. FIG. 3A provides a schematic of a bi-cistronic vector for driving the expression of a synthetic crRNA-tracrRNA chimera (chimeric RNA) as well as SpCas9. The chimeric guide RNA contains a 20-bp guide sequence corresponding to the protospacer in the genomic target site. FIG. 3B provides a schematic showing guide sequences targeting the human EMX1, PVALB, and mouse Th loci, as well as their predicted secondary structures. The modification efficiency at each target site is indicated below the RNA secondary structure drawing (EMX1, n=216 amplicon sequencing reads; PVALB, n=224 reads; Th, n=265 reads). The folding algorithm produced an output with each base colored according to its probability of assuming the predicted secondary structure, as indicated by a rainbow scale that is reproduced in FIG. 3B in gray scale. Further vector designs for SpCas9 are shown in FIG. 3A, including single expression vectors incorporating a U6 promoter linked to an insertion site for a guide oligo, and a Cbh promoter linked to SpCas9 coding sequence.
[0165] To test whether spacers containing secondary structures are able to function in prokaryotic cells where CRISPRs naturally operate, transformation interference of protospacer-bearing plasmids were tested in an E. coli strain heterologously expressing the S. pyogenes SF370 CRISPR locus 1 (FIG. 3C). The CRISPR locus was cloned into a low-copy E. coli expression vector and the crRNA array was replaced with a single spacer flanked by a pair of DRs (pCRISPR). E. coli strains harboring different pCRISPR plasmids were transformed with challenge plasmids containing the corresponding protospacer and PAM sequences (FIG. 3C). In the bacterial assay, all spacers facilitated efficient CRISPR interference (FIG. 3C). These results suggest that there may be additional factors affecting the efficiency of CRISPR activity in mammalian cells.
[0166] To investigate the specificity of CRISPR-mediated cleavage, the effect of single-nucleotide mutations in the guide sequence on protospacer cleavage in the mammalian genome was analyzed using a series of EMX1-targeting chimeric crRNAs with single point mutations (FIG. 4A). FIG. 4B illustrates results of a Surveyor nuclease assay comparing the cleavage efficiency of Cas9 when paired with different mutant chimeric RNAs. Single-base mismatch up to 12-bp 5' of the PAM substantially abrogated genomic cleavage by SpCas9, whereas spacers with mutations at farther upstream positions retained activity against the original protospacer target (FIG. 4B). In addition to the PAM, SpCas9 has single-base specificity within the last 12-bp of the spacer. Furthermore, CRISPR is able to mediate genomic cleavage as efficiently as a pair of TALE nucleases (TALEN) targeting the same EMX1 protospacer. FIG. 4C provides a schematic showing the design of TALENs targeting E EMX1, and FIG. 4D shows a Surveyor gel comparing the efficiency of TALEN and Cas9 (n=3).
[0167] Having established a set of components for achieving CRISPR-mediated gene editing in mammalian cells through the error-prone NHEJ mechanism, the ability of CRISPR to stimulate homologous recombination (HR), a high fidelity gene repair pathway for making precise edits in the genome, was tested. The wild type SpCas9 is able to mediate site-specific DSBs, which can be repaired through both NHEJ and HR. In addition, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of SpCas9 was engineered to convert the nuclease into a nickase (SpCas9n; illustrated in FIG. 5A) (see e.g. Sapranausaks et al., 2011, Cucleic Acis Research, 39: 9275; Gasiunas et al., 2012, Proc. Natl. Acad. Sci. USA, 109:E2579), such that nicked genomic DNA undergoes the high-fidelity homology-directed repair (HDR). Surveyor assay confirmed that SpCas9n does not generate indels at the EMX1 protospacer target. As illustrated in FIG. 5B, co-expression of EMX1-targeting chimeric crRNA with SpCas9 produced indels in the target site, whereas co-expression with SpCas9n did not (n=3). Moreover, sequencing of 327 amplicons did not detect any indels induced by SpCas9n. The same locus was selected to test CRISPR-mediated HR by co-transfecting HEK 293FT cells with the chimeric RNA targeting EMX1, hSpCas9 or hSpCas9n, as well as a HR template to introduce a pair of restriction sites (HindIII and NheI) near the protospacer. FIG. 5C provides a schematic illustration of the HR strategy, with relative locations of recombination points and primer annealing sequences (arrows). SpCas9 and SpCas9n indeed catalyzed integration of the HR template into the EMX1 locus. PCR amplification of the target region followed by restriction digest with HindIII revealed cleavage products corresponding to expected fragment sizes (arrows in restriction fragment length polymorphism gel analysis shown in FIG. 5D), with SpCas9 and SpCas9n mediating similar levels of HR efficiencies. Applicants further verified HR using Sanger sequencing of genomic amplicons (FIG. 5E). These results demonstrate the utility of CRISPR for facilitating targeted gene insertion in the mammalian genome. Given the 14-bp (12-bp from the spacer and 2-bp from the PAM) target specificity of the wild type SpCas9, the availability of a nickase can significantly reduce the likelihood of off-target modifications, since single strand breaks are not substrates for the error-prone NHEJ pathway.
[0168] Expression constructs mimicking the natural architecture of CRISPR loci with arrayed spacers (FIG. 2A) were constructed to test the possibility of multiplexed sequence targeting. Using a single CRISPR array encoding a pair of EMX1- and PVALB-targeting spacers, efficient cleavage at both loci was detected (FIG. 4F, showing both a schematic design of the crRNA array and a Surveyor blot showing efficient mediation of cleavage). Targeted deletion of larger genomic regions through concurrent DSBs using spacers against two targets within EMX1 spaced by 119 bp was also tested, and a 1.6% deletion efficacy (3 out of 182 amplicons; FIG. 5G) was detected. This demonstrates that the CRISPR system can mediate multiplexed editing within a single genome.
Example 2
CRISPR System Modifications and Alternatives
[0169] The ability to use RNA to program sequence-specific DNA cleavage defines a new class of genome engineering tools for a variety of research and industrial applications. Several aspects of the CRISPR system can be further improved to increase the efficiency and versatility of CRISPR targeting. Optimal Cas9 activity may depend on the availability of free Mg2+ at levels higher than that present in the mammalian nucleus (see e.g. Jinek et al., 2012. Science, 337:816), and the preference for an NGG motif immediately downstream of the protospacer restricts the ability to target on average every 12-bp in the human genome. Some of these constraints can be overcome by exploring the diversity of CRISPR loci across the microbial metagenome (see e.g. Makarova et al., 2011, Nat Rev Microbiol, 9:467). Other CRISPR loci may be transplanted into the mammalian cellular milieu by a process similar to that described in Example 1. The modification efficiency at each target site is indicated below the RNA secondary structures. The algorithm generating the structures colors each base according to its probability of assuming the predicted secondary structure. RNA guide spacers 1 and 2 induced 14% and 6.4%, respectively. Statistical analysis of cleavage activity across biological replica at these two protospacer sites is also provided in FIG. 7.
Example 3
Sample Target Sequence Selection Algorithm
[0170] A software program is designed to identify candidate CRISPR target sequences on both strands of an input DNA sequence based on desired guide sequence length and a CRISPR motif sequence (PAM) for a specified CRISPR enzyme. For example, target sites for Cas9 from S. pyogenes, with PAM sequences NGG, may be identified by searching for 5'-Nx-NGG-3' both on the input sequence and on the reverse-complement of the input. Likewise, target sites for Cas9 of S. thermophilus CRISPR1, with PAM sequence NNAGAAW, may be identified by searching for 5'-Nx-NNAGAAW-3' both on the input sequence and on the reverse-complement of the input. Likewise, target sites for Cas9 of S. thermophilus CRISPR3, with PAM sequence NGGNG, may be identified by searching for 5'-N,-NGGNG-3' both on the input sequence and on the reverse-complement of the input. The value "x" in Nx may be fixed by the program or specified by the user, such as 20.
[0171] Since multiple occurrences in the genome of the DNA target site may lead to nonspecific genome editing, after identifying all potential sites, the program filters out sequences based on the number of times they appear in the relevant reference genome. For those CRISPR enzymes for which sequence specificity is determined by a `seed` sequence, such as the 11-12 bp 5' from the PAM sequence, including the PAM sequence itself, the filtering step may be based on the seed sequence. Thus, to avoid editing at additional genomic loci, results are filtered based on the number of occurrences of the seed:PAM sequence in the relevant genome. The user may be allowed to choose the length of the seed sequence. The user may also be allowed to specify the number of occurrences of the seed:PAM sequence in a genome for purposes of passing the filter. The default is to screen for unique sequences. Filtration level is altered by changing both the length of the seed sequence and the number of occurrences of the sequence in the genome. The program may in addition or alternatively provide the sequence of a guide sequence complementary to the reported target sequence(s) by providing the reverse complement of the identified target sequence(s).
[0172] Further details of methods and algorithms to optimize sequence selection can be found found in U.S. application Ser. No. TBA (Broad Reference BI-2012/084 44790.11.2022); incorporated herein by reference.
Example 4
Evaluation of Multiple Chimeric crRNA-tracrRNA Hybrids
[0173] This example describes results obtained for chimeric RNAs (chiRNAs; comprising a guide sequence, a tracr mate sequence, and a tracr sequence in a single transcript) having tracr sequences that incorporate different lengths of wild-type tracrRNA sequence. FIG. 18a illustrates a schematic of a bicistronic expression vector for chimeric RNA and Cas9. Cas9 is driven by the CBh promoter and the chimeric RNA is driven by a U6 promoter. The chimeric guide RNA consists of a 20 bp guide sequence (Ns) joined to the tracr sequence (running from the first "U" of the lower strand to the end of the transcript), which is truncated at various positions as indicated. The guide and tracr sequences are separated by the tracr-mate sequence GUUUUAGAGCUA followed by the loop sequence GAAA. Results of SURVEYOR assays for Cas9-mediated indels at the human EMX1 and PVALB loci are illustrated in FIGS. 18b and 18c, respectively. Arrows indicate the expected SURVEYOR fragments. ChiRNAs are indicated by their "+n" designation, and crRNA refers to a hybrid RNA where guide and tracr sequences are expressed as separate transcripts. Quantification of these results, performed in triplicate, are illustrated by histogram in FIGS. 11a and 11b, corresponding to FIGS. 10b and 10c, respectively ("N.D." indicates no indels detected). Protospacer IDs and their corresponding genomic target, protospacer sequence, PAM sequence, and strand location are provided in Table D. Guide sequences were designed to be complementary to the entire protospacer sequence in the case of separate transcripts in the hybrid system, or only to the underlined portion in the case of chimeric RNAs.
TABLE-US-00004 TABLE D protospacer genomic ID target protospacer sequence (5' to 3') PAM Strand 1 EMX1 GGACATCGATGTCACCTCCAATGACTAG TGG + GG 2 EMX1 CATTGGAGGTGACATCGATGTCCTCCCC TGG - AT 3 EMX1 GGAAGGGCCTGAGTCCGAGCAGAAGAA GGG + GAA 4 PVALB GGTGGCGAGAGGGGCCGAGATTGGGTGT AGG + TC 5 PVALB ATGCAGGAGGGTGGCGAGAGGGGCCGA TGG + GAT
Cell Culture and Transfection
[0174] Human embryonic kidney (HEK) cell line 293FT (Life Technologies) was maintained in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100U/mL penicillin, and 100 μg/mL streptomycin at 37° C. with 5% CO2 incubation. 293FT cells were seeded onto 24-well plates (Corning) 24 hours prior to transfection at a density of 150,000 cells per well. Cells were transfected using Lipofectamine 2000 (Life Technologies) following the manufacturer's recommended protocol. For each well of a 24-well plate, a total of 500 ng plasmid was used.
SURVEYOR Assay for Genome Modification
[0175] 293FT cells were transfected with plasmid DNA as described above. Cells were incubated at 37° C. for 72 hours post-transfection prior to genomic DNA extraction. Genomic DNA was extracted using the QuickExtract DNA Extraction Solution (Epicentre) following the manufacturer's protocol. Briefly, pelleted cells were resuspended in QuickExtract solution and incubated at 65° C. for 15 minutes and 98° C. for 10 minutes. The genomic region flanking the CRISPR target site for each gene was PCR amplified (primers listed in Table E), and products were purified using QiaQuick Spin Column (Qiagen) following the manufacturer's protocol. 400 ng total of the purified PCR products were mixed with 2 μl 10×Taq DNA Polymerase PCR buffer (Enzymatics) and ultrapure water to a final volume of 20 μl, and subjected to a re-annealing process to enable heteroduplex formation: 95° C. for 10 min, 95° C. to 85° C. ramping at -2° C./s. 85° C. to 25° C. at -0.25° C./s, and 25° C. hold for 1 minute. After re-annealing, products were treated with SURVEYOR nuclease and SURVEYOR enhancer S (Transgenomics) following the manufacturer's recommended protocol, and analyzed on 4-20% Novex TBE poly-acrylamide gels (Life Technologies). Gels were stained with SYBR Gold DNA stain (Life Technologies) for 30 minutes and imaged with a Gel Doc gel imaging system (Bio-rad). Quantification was based on relative band intensities.
TABLE-US-00005 TABLE E genomic primer name target primer sequence (5' to 3') Sp-EMX1-F EMX1 AAAACCACCCTTCTCTCTGGC Sp-EMX1-R EMX1 GGAGATTGGAGACACGGAGAG Sp-PVALB-F PVALB CTGGAAAGCCAATGCCTGAC Sp-PVALB-R PVALB GGCAGCAAACTCCTTGTCCT
Computational Identification of Unique CRISPR Target Sites
[0176] To identify unique target sites for the S. pyogenes SF370 Cas9 (SpCas9) enzyme in the human, mouse, rat, zebrafish, fruit fly, and C. elegans genome, we developed a software package to scan both strands of a DNA sequence and identify all possible SpCas9 target sites. For this example, each SpCas9 target site was operationally defined as a 20 bp sequence followed by an NGG protospacer adjacent motif (PAM) sequence, and we identified all sequences satisfying this 5'-N20-NGG-3' definition on all chromosomes. To prevent non-specific genome editing, after identifying all potential sites, all target sites were filtered based on the number of times they appear in the relevant reference genome. To take advantage of sequence specificity of Cas9 activity conferred by a `seed` sequence, which can be, for example, approximately 11-12 bp sequence 5' from the PAM sequence, 5'-NNNNNNNNNN-NGG-3' sequences were selected to be unique in the relevant genome. All genomic sequences were downloaded from the UCSC Genome Browser (Human genome hg19, Mouse genome mm9, Rat genome rn5, Zebrafish genome danRer7, D. melanogaster genome dm4 and C. elegans genome ce10). The full search results are available to browse using UCSC Genome Browser information. An example visualization of some target sites in the human genome is provided in FIG. 22.
[0177] Initially, three sites within the EMX1 locus in human HEK 293FT cells were targeted. Genome modification efficiency of each chiRNA was assessed using the SURVEYOR nuclease assay, which detects mutations resulting from DNA double-strand breaks (DSBs) and their subsequent repair by the non-homologous end joining (NHEJ) DNA damage repair pathway. Constructs designated chiRNA(+n) indicate that up to the +n nucleotide of wild-type tracrRNA is included in the chimeric RNA construct, with values of 48, 54, 67, and 85 used for n. Chimeric RNAs containing longer fragments of wild-type tracrRNA (chiRNA(+67) and chiRNA(+85)) mediated DNA cleavage at all three EMX1 target sites, with chiRNA(+85) in particular demonstrating significantly higher levels of DNA cleavage than the corresponding crRNA/tracrRNA hybrids that expressed guide and tracr sequences in separate transcripts (FIGS. 10b and 10a). Two sites in the PVALB locus that yielded no detectable cleavage using the hybrid system (guide sequence and tracr sequence expressed as separate transcripts) were also targeted using chiRNAs. chiRNA(+67) and chiRNA(+85) were able to mediate significant cleavage at the two PVALB protospacers (FIGS. 10c and 10b).
[0178] For all five targets in the EMX1 and PVALB loci, a consistent increase in genome modification efficiency with increasing tracr sequence length was observed. Without wishing to be bound by any theory, the secondary structure formed by the 3' end of the tracrRNA may play a role in enhancing the rate of CRISPR complex formation. An illustration of predicted secondary structures for each of the chimeric RNAs used in this example is provided in FIG. 21. The secondary structure was predicted using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) using minimum free energy and partition function algorithm. Pseudocolor for each based (reproduced in grayscale) indicates the probability of pairing. Because chiRNAs with longer tracr sequences were able to cleave targets that were not cleaved by native CRISPR crRNA/tracrRNA hybrids, it is possible that chimeric RNA may be loaded onto Cas9 more efficiently than its native hybrid counterpart. To facilitate the application of Cas9 for site-specific genome editing in eukaryotic cells and organisms, all predicted unique target sites for the S. pyogenes Cas9 were computationally identified in the human, mouse, rat, zebra fish, C. elegans, and D. melanogaster genomes. Chimeric RNAs can be designed for Cas9 enzymes from other microbes to expand the target space of CRISPR RNA-programmable nucleases.
[0179] FIGS. 11 and 21 illustrate exemplary bicistronic expression vectors for expression of chimeric RNA including up to the +85 nucleotide of wild-type tracr RNA sequence, and SpCas9 with nuclear localization sequences. SpCas9 is expressed from a CBh promoter and terminated with the bGH polyA signal (bGH pA). The expanded sequence illustrated immediately below the schematic corresponds to the region surrounding the guide sequence insertion site, and includes, from 5' to 3',3'-portion of the U6 promoter (first shaded region), BbsI cleavage sites (arrows), partial direct repeat (tracr mate sequence GTTTTAGAGCTA, underlined), loop sequence GAAA, and +85 tracr sequence (underlined sequence following loop sequence). An exemplary guide sequence insert is illustrated below the guide sequence insertion site, with nucleotides of the guide sequence for a selected target represented by an "N".
[0180] Sequences described in the above examples are as follows (polynucleotide sequences are 5' to 3'):
[0181] U6-short tracrRNA (Streptococcus pyogenes SF370):
TABLE-US-00006 GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGC TGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAG TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTT TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAA GTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG GAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCA ACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (bold = tracrRNA sequence; underline = terminator sequence)
[0182] U6-long tracrRNA (Streptococcus pyogenes SF370):
TABLE-US-00007 GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGC TGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAG TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTT TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAA GTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG GTAGTATTAAGTATTGTTTTATGGCTGATAAATTTCTTTGAATTTCTCCT TGATTATTTGTTATAAAAGTTATAAAATAATCTTGTTGGAACCATTCAAA ACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGCTTTTTTT
[0183] U6-DR-BbsI backbone-DR (Streptococcus pyogenes SF370):
TABLE-US-00008 GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGC TGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAG TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTT TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAA GTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG GGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACGGGTCTTCGAGAA GACGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC
[0184] U6-chimeric RNA-BbsI backbone (Streptococcus pyogenes SF370)
TABLE-US-00009 GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGC TGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAG TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTT TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAA GTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG GGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG GCTAGTCCG
[0185] NLS-SpCas9-EGFP:
TABLE-US-00010 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDI GTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDK KHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI LSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAED AKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTE ITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKA QVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKLEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSE LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVY GDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKR PLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESI LPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGDAAAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATY GKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMP EGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH KLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIG DGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
[0186] SpCas9-EGFP-NLS:
TABLE-US-00011 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDAAAVSKGEELFTGVVPILVELDGDVNGHKFSV SGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMK QHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGID FKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGI TLGMDELYKKRPAATKKAGQAKKKK
[0187] NLS-SpCas9-EGFP-NLS:
TABLE-US-00012 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDI GTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDK KHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI LSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAED AKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTE ITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG YTDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKA QVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSD KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEL DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEEVYG DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGDAAAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYG KLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPE GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK LEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGD GPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKR PAATKKAGQAKKKK
[0188] NLS-SpCas9-NLS:
TABLE-US-00013 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDI GTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDK KHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI LSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAED AKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTE ITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIP HQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKA QVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSD KNRGKSDNVPSEEVVKMKNYWQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLV SDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPK RNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKLR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL SQLGGDKRPAATKKAGQAKKKK
[0189] NLS-mCherry-SpRNase3:
TABLE-US-00014 MFLFLSLTSFLSSSRTLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEI EGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADI PDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNF PSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKT TYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDEL YKGSKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVSHNERLE FLGDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGFSRFCSFD AYIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVRRFLKQVMI PQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPAHAKQFEVSI VVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEV
[0190] SpRNase3-mCherry-NLS:
TABLE-US-00015 MKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVSHNERLEFLG DAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGFSRFCSFDAYI KLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVRRFLKQVMIPQV EKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPAHAKQFEVSIVVN GAVLSKGLGKSKKLAEQDAAKNALAQLSEVGSVSKGEEDNMAIIKEFMRF KVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQ FMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQ DGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQR LKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQY ERAEGRHSTGGMDELYKKRPAATKKAGQAKKKK
[0191] NLS-SpCas9n-NLS (the D10A nickase mutation is lowercase):
TABLE-US-00016 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLaI GTNSVGWAVITDEYKWSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPH QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL SQLGGDKRPAATKKAGQAKKKK
[0192] hEMX1-HR Template-HindII-NheI:
TABLE-US-00017 GAATGCTGCCCTCAGACCCGCTTCCTCCCTGTCCTTGTCTGTCCAAGGAG AATGAGGTCTCACTGGTGGATTTCGGACTACCCTGAGGAGCTGGCACCTG AGGGACAAGGCCCCCCACCTGCCCAGCTCCAGCCTCTGATGAGGGGTGGG AGAGAGCTACATGAGGTTGCTAAGAAAGCCTCCCCTGAAGGAGACCACAC AGTGTGTGAGGTTGGAGTCTCTAGCAGCGGGTTCTGTGCCCCCAGGGATA GTCTGGCTGTCCAGGCACTGCTCTTGATAAAACACCACCTCCTAGTTATG AAACCATGCCCATTCTGCCTCTCTGTATGGAAAAGAGCATGGGGCTGGCC CGTGGGGTGGTGTCCACTTTAGGCCCTGTGGGAGATCATGGGAACCCACG CAGTGGGTCATAGGCTCTCTCATTTACTACTCACATCCACTCTGTGAAGA AGCGATTATGATCTCTCCTCTAGAAACTCGTAGAGTCCCATGTCTGCCGG CTTCCAGAGCCTGCACTCCTCCACCTTGGCTTGGCTTTGCTGGGGCTAGA GGAGCTAGGATGCACAGCAGCTCTGTGACCCTTTGTTTGAGAGGAACAGG AAAACCACCCTTCTCTCTGGCCCACTGTGTCCTCTTCCTGCCCTGCCATC CCCTTCTGTGAATGTTAGACCCATGGGAGCAGCTGGTCAGAGGGGACCCC GGCCTGGGGCCCCTAACCCTATGTAGCCTCAGTCTTCCCATCAGGCTCTC AGCTCAGCCTGAGTGTTGAGGCCCCAGTGGCTGCTCTGGGGGCCTCCTGA GTTTCTCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCC AGAACCGGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCT GAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCAT TGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACa agcttgctagcGGTGGGCAACCACAAACCCACGAGGGCAGAGTGCTGCTT GCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCCACTCCC TGGCCAGGCTTTGGGGAGGCCTGGAGTCATGGCCCCACAGGGCTTGAAGC CCGGGGCCGCCATTGACAGAGGGACAAGCAATGGGCTGGCTGAGGCCTGG GACCACTTGGCCTTCTCCTCGGAGAGCCTGCCTGCCTGGGCGGGCCCGCC CGCCACCGCAGCCTCCCAGCTGCTCTCCGTGTCTCCAATCTCCCTTTTGT TTTGATGCATTTCTGTTTTAATTTATTTTCCAGGCACCACTGTAGTTTAG TGATCCCCAGTGTCCCCCTTCCCTATGGGAATAATAAAAGTCTCTCTCTT AATGACACGGGCATCCAGCTCCAGCCCCAGAGCCTGGGGTGGTAGATTCC GGCTCTGAGGGCCAGTGGGGGCTGGTAGAGCAAACGCGTTCAGGGCCTGG GAGCCTGGGGTGGGGTACTGGTGGAGGGGGTCAAGGGTAATTCATTAACT CCTCTCTTTTGTTGGGGGACCCTGGTCTCTACCTCCAGCTCCACAGCAGG AGAAACAGGCTAGACATAGGGAAGGGCCATCCTGTATCTTGAGGGAGGAC AGGCCCAGGTCTTTCTTAACGTATTGAGAGGTGGGAATCAGGCCCAGGTA GTTCAATGGGAGAGGGAGAGTGCTTCCCTCTGCCTAGAGACTCTGGTGGC TTCTCCAGTTGAGGAGAAACCAGAGGAAAGGGGAGGATTGGGGTCTGGGG GAGGGAACACCATTCACAAAGGCTGACGGTTCCAGTCCGAAGTCGTGGGC CCACCAGGATGCTCACCTGTCCTTGGAGAACCGCTGGGCAGGTTGAGACT GCAGAGACAGGGCTTAAGGCTGAGCCTGCAACCAGTCCCCAGTGACTCAG GGCCTCCTCAGCCCAAGAAAGAGCAACGTGCCAGGGCCCGCTGAGCTCTT GTGTTCACCTG
[0193] NLS-StCsn1-NLS:
TABLE-US-00018 MKRPAATKKAGQAKKKKSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRI FPAAQAENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISI NLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGD YAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVF PTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKS RTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNLLNDL NNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADI KGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTE REGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMME LIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAK SVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANKDEK DAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTIS IHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQAL DSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNL VDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDT YHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYK ESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRQAK VGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEK VIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKS LKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLK YADLQFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDT ETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQ CKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDFKRPAATKKAGQAK KKK
[0194] U6-St_tracrRNA(7-97):
TABLE-US-00019 GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGC TGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAG TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTT TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAA GTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG TTACTTAAATCTTGCAGAAGCTACAAAGATAAGGCTTCATGCCGAAATCA ACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTAA
[0195] U6-DR-spacer-DR (S. pyogenes SF370)
TABLE-US-00020 gagggcctatttcccatgattccttcatatttgcatatacgatacaaggc tgttagagagataattggaattaatttgactgtaaacacaaaagatatta gtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagt tttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaa agtatttcgatttcttggctttatatatcttgtggaaaggacgaaacacc gggttttagagctatgctgttttgaatggtcccaaaacNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNgttttagagctatgctgttttgaatggtccca aaacTTTTTTT (lowercase underline = direct repeat; N = guide sequence; bold = terminator)
[0196] Chimeric RNA containing+48 tracr RNA (S. pyogenes SF370)
TABLE-US-00021 gagggcctatttcccatgattccttcatatttgcatatacgatacaaggc tgttagagagataattggaattaatttgactgtaaacacaaagatattag tacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaa gtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccN NNNNNNNNNNNNNNNNNNNgttttgaggctagaaatagcaagttaaaata aggctagtccgTTTTTTT (N = guide sequence; first underline = tracr matesequence; second underline = tracr sequence; bold = terminator)
[0197] Chimeric RNA containing+54 tracr RNA (S. pyogenes SF370)
TABLE-US-00022 gagggcctatttcccatgattccttcatatttgcatatacgatacaaggc tgttagagagataattggaattaatttgactgtaaacacaaagatattag tacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaa gtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccN NNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaata aggctagtccgttatcaTTTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)
[0198] Chimeric RNA containing+67 tracr RNA (S. pyogenes SF370)
TABLE-US-00023 gagggcctatttcccatgattccttcatatttgcatatacgatacaaggc tgttagagagataattggaattaatttgactgtaaacacaaagatattag tacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaa gtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccN NNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaata aggctagtccgttatcaacttgaaaaagtgTTTTTTT (N = guide sequence; firstunderline = tracr mate sequence; second underline = tracr sequence; bold = terminator)
[0199] Chimeric RNA containing+85 tracr RNA (S. pyogenes SF370)
TABLE-US-00024 gagggcctatttcccatgattccttcatatttgcatatacgatacaaggc tgttagagagataattggaattaatttgactgtaaacacaaagatattag tacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaa gtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccN NNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaata aggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTT TT (N = guidesequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)
[0200] CBh-NLS-SpCas9-NLS
TABLE-US-00025 CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCA CTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACG TAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTAT GGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC ATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCC CTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAG CGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCA GAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGC GGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTG CCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGC TCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCT CCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTT GGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCA CTTTTTTTCAGGTTGGaccggtgccaccATGGACTATAAGGACCACGACG GAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATG GCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGA CAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGG CCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTG GGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCT GTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCA GAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATC TTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGA AGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCT TCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATC TACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCG GCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCC TGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTC ATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAA CGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGA GCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAAT GGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTT CAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGG ACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAG TACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCT GAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCG CCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTG AAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTT CGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCC AGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGC ACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCA GCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGC TGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGAC AACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGT GGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGA GCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGC GCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCT GCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA CCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGA AAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCT GTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACT TCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGAT CGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAA GGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATA TCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGG CTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAA GCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACG GCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCC GACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCT GACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATA GCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAG GGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGG CCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGA CCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAA GAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGA AAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATG GGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGAC TACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCAT CGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACA ACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAG CTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAA GGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGA GACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTG GACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGA AGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGG ATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCAC GACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCC TAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGC GGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAG TACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCT GGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAA CCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAA GTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGAC AGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGC TGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGAC AGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGG CAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCA TGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAG GGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTC CCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCG AACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTC CTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAA TGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGA TCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCT AATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCAT CAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGG GAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGG TACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCAT CACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACT TTCTTTTTCTTAGCTTGACCAGCTTTCTTAGTAGCAGCAGGACGCTTTAA (underline = NLS-hSpCas9-NLS)
[0201] Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW)
TABLE-US-00026 NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaa tcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgt cattttatggcagggtgttttcgttatttaaTTTTTT (N = guide sequence; firstunderline = tracr mate sequence; second underline = tracr sequence; bold = terminator)
[0202] Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW)
TABLE-US-00027 NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagcta caaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgttttcgttatttaaTTTTTT (N = guide sequence; first underline = tracrmate sequence; second underline = tracr sequence; bold = terminator)
[0203] Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW)
TABLE-US-00028 NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagcta caaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgtTTTTTT (N = guide sequence; first underline = tracr mate sequence;second underline = tracr sequence; bold = terminator)
[0204] Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW)
TABLE-US-00029 NNNNNNNNNNNNNNNNNNNNgttattgtactctcaagatttaGAAAtaaa tcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgt cattttatggcagggtgttttcgttatttaaTTTTTT (N = guide sequence; firstunderline = tracr mate sequence; second underline = tracr sequence; bold = terminator)
[0205] Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW)
TABLE-US-00030 NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagcta caaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgttttcgttatttaaTTTTTT (N = guide sequence; first underline = tracrmate sequence; second underline = tracr sequence; bold = terminator)
[0206] Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW)
TABLE-US-00031 NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagcta caaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgtTTTTTT (N = guide sequence; first underline = tracr matesequence; second underline = tracr sequence; bold = terminator)
[0207] Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW)
TABLE-US-00032 NNNNNNNNNNNNNNNNNNNNgttattgtactctcaagatttaGAAAtaaa tcttgcagaagctacaatgataaggcttcatgccgaaatcaacaccctgt cattttatggcagggtgttttcgttatttaaTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator)
[0208] Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW)
TABLE-US-00033 NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagcta caatgataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgttttcgttatttaaTTTTTT (N = guide sequence; first underline = tracrmate sequence; second underline = tracr sequence; bold = terminator)
[0209] Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW)
TABLE-US-00034 NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagcta caatgataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgtTTTTTT (N = guide sequence; first underline = tracr mate sequence;second underline = tracr sequence; bold = terminator)
[0210] Example chimeric RNA for S. thermophilus LMD-9 CRISPR3 Cas9 (with PAM of NGGNG)
TABLE-US-00035 NNNNNNNNNNNNNNNNNNNNgttttagagctgtgGAAAcacagcgagtta aaataaggcttagtccgtactcaacttgaaaaggtggcaccgattcggt gtTTTTTT (N = guide sequence; first underline = tracr mate sequence;second underline = tracr sequence; bold = terminator)
[0211] Codon-optimized version of Cas9 from S. thermophilus LMD-9 CRISPR3 locus (with an NLS at both 5' and 3' ends)
TABLE-US-00036 ATGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAA GACCAAGCCCTACAGCATCGGCCTGGACATCGGCACCAATAGCGTGGGCT GGGCCGTGACCACCGACAACTACAAGGTGCCCAGCAAGAAAATGAAGGTG CTGGGCAACACCTCCAAGAAGTACATCAAGAAAAACCTGCTGGGCGTGCT GCTGTTCGACAGCGGCATTACAGCCGAGGGCAGACGGCTGAAGAGAACCG CCAGACGGCGGTACACCCGGCGGAGAAACAGAATCCTGTATCTGCAAGAG ATCTTCAGCACCGAGATGGCTACCCTGGACGACGCCTTCTTCCAGCGGCT GGACGACAGCTTCCTGGTGCCCGACGACAAGCGGGACAGCAAGTACCCCA TCTTCGGCAACCTGGTGGAAGAGAAGGCCTACCACGACGAGTTCCCCACC ATCTACCACCTGAGAAAGTACCTGGCCGACAGCACCAAGAAGGCCGACCT GAGACTGGTGTATCTGGCCCTGGCCCACATGATCAAGTACCGGGGCCACT TCCTGATCGAGGGCGAGTTCAACAGCAAGAACAACGACATCCAGAAGAAC TTCCAGGACTTCCTGGACACCTACAACGCCATCTTCGAGAGCGACCTGTC CCTGGAAAACAGCAAGCAGCTGGAAGAGATCGTGAAGGACAAGATCAGCA AGCTGGAAAAGAAGGACCGCATCCTGAAGCTGTTCCCCGGCGAGAAGAAC AGCGGAATCTTCAGCGAGTTTCTGAAGCTGATCGTGGGCAACCAGGCCGA CTTCAGAAAGTGCTTCAACCTGGACGAGAAAGCCAGCCTGCACTTCAGCA AAGAGAGCTACGACGAGGACCTGGAAACCCTGCTGGGATATATCGGCGAC GACTACAGCGACGTGTTCCTGAAGGCCAAGAAGCTGTACGACGCTATCCT GCTGAGCGGCTTCCTGACCGTGACCGACAACGAGACAGAGGCCCCACTGA GCAGCGCCATGATTAAGCGGTACAACGAGCACAAAGAGGATCTGGCTCTG CTGAAAGAGTACATCCGGAACATCAGCCTGAAAACCTACAATGAGGTGTT CAAGGACGACACCAAGAACGGCTACGCCGGCTACATCGACGGCAAGACCA ACCAGGAAGATTTCTATGTGTACCTGAAGAAGCTGCTGGCCGAGTTCGAG GGGGCCGACTACTTTCTGGAAAAAATCGACCGCGAGGATTTCCTGCGGAA GCAGCGGACCTTCGACAACGGCAGCATCCCCTACCAGATCCATCTGCAGG AAATGCGGGCCATCCTGGACAAGCAGGCCAAGTTCTACCCATTCCTGGCC AAGAACAAAGAGCGGATCGAGAAGATCCTGACCTTCCGCATCCCTACTAC GTGGGCCCCCTGGCCAGAGGCAACAGCGATTTTGCCTGGTCCATCCGGAA GCGCAATGAGAAGATCACCCCCTGGAACTTCGAGGACGTGATCGACAAAG AGTCCAGCGCCGAGGCCTTCATCAACCGGATGACCAGCTTCGACCTGTAC CTGCCCGAGGAAAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGACATT CAATGTGTATAACGAGCTGACCAAAGTGCGGTTTATCGCCGAGTCTATGC GGGACTACCAGTTCCTGGACTCCAAGCAGAAAAAGGACATCGTGCGGCTG TACTTCAAGGACAAGCGGAAAGTGACCGATAAGGACATCATCGAGTACCT GCACGCCATCTACGGCTACGATGGCATCGAGCTGAAGGGCATCGAGAAGC AGTTCAACTCCAGCCTGAGCACATACCACGACCTGCTGAACATTATCAAC GACAAAGAATTTCTGGACGACTCCAGCAACGAGGCCATCATCGAAGAGAT CATCCACACCCTGACCATCTTTGAGGACCGCGAGATGATCAAGCAGCGGC TGAGCAAGTTCGAGAACATCTTCGACAAGAGCGTGCTGAAAAAGCTGAGC AGACGGCACTACACCGGCTGGGGCAAGCTGAGCGCCAAGCTGATCAACGG CATCCGGGACGAGAAGTCCGGCAACACAATCCTGGACTACCTGATCGACG ACGGCATCAGCAACCGGAACTTCATGCAGCTGATCCACGACGACGCCCTG AGCTTCAAGAAGAAGATCCAGAAGGCCCAGATCATCGGGGACGAGGACAA GGGCAACATCAAAGAAGTCGTGAAGTCCCTGCCCGGCAGCCCCGCCATCA AGAAGGGAATCCTGCAGAGCATCAAGATCGTGGACGAGCTCGTGAAAGTG ATGGGCGGCAGAAAGCCCGAGAGCATCGTGGTGGAAATGGCTAGAGAGAA CCAGTACACCAATCAGGGCAAGAGCAACAGCCAGCAGAGACTGAAGAGAC TGGAAAAGTCCCTGAAAGAGCTGGGCAGCAAGATTCTGAAAGAGAATATC CCTGCCAAGCTGTCCAAGATCGACAACAACGCCCTGCAGAACGACCGGCT GTACCTGTACTACCTGCAGAATGGCAAGGACATGTATACAGGCGACGACC TGGATATCGACCGCCTGAGCAACTACGACATCGACCATATTATCCCCCAG GCCTTCCTGAAAGACAACAGCATTGACAACAAAGTGCTGGTGTCCTCCGC CAGCAACCGCGGCAAGTCCGATGATGTGCCCAGCCTGGAAGTCGTGAAAA AGAGAAAGACCTTCTGGTATCAGCTGCTGAAAAGCAAGCTGATTAGCCAG AGGAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCCCTGA AGATAAGGCCGGCTTCATCCAGAGACAGCTGGTGGAAACCCGGCAGATCA CCAAGCACGTGGCCAGACTGCTGGATGAGAAGTTTAACAACAAGAAGGAC GAGAACAACCGGGCCGTGCGGACCGTGAAGATCATCACCCTGAAGTCCAC CCTGGTGTCCCAGTTCCGGAAGGACTTCGAGCTGTATAAAGTGCGCGAGA TCAATGACTTTCACCACGCCCACGACGCCTACCTGAATGCCGTGGTGGCT TCCGCCCTGCTGAAGAAGTACCCTAAGCTGGAACCCGAGTTCGTGTACGG CGACTACCCCAAGTACAACTCCTTCAGAGAGCGGAAGTCCGCCACCGAGA AGGTGTACTTCTACTCCAACATCATGAATATCTTTAAGAAGTCCATCTCC CTGGCCGATGGCAGAGTGATCGAGCGGCCCCTGATCGAAGTGAACGAAGA GACAGGCGAGAGCGTGTGGAACAAAGAAAGCGACCTGGCCACCGTGCGGC GGGTGCTGAGTTATCCTCAAGTGAATGTCGTGAAGAAGGTGGAAGAACAG AACCACGGCCTGGATCGGGGCAAGCCCAAGGGCCTGTTCAACGCCAACCT GTCCAGCAAGCCTAAGCCCAACTCCAACGAGAATCTCGTGGGGGCCAAAG AGTACCTGGACCCTAAGAAGTACGGCGGATACGCCGGCATCTCCAATAGC TTCACCGTGCTCGTGAAGGGCACAATCGAGAAGGGCGCTAAGAAAAAGAT CACAAACGTGCTGGAATTTCAGGGGATCTCTATCCTGGACCGGATCAACT ACCGGAAGGATAAGCTGAACTTTCTGCTGGAAAAAGGCTACAAGGACATT GAGCTGATTATCGAGCTGCCTAAGTACTCCCTGTTCGAACTGAGCGACGG CTCCAGACGGATGCTGGCCTCCATCCTGTCCACCAACAACAAGCGGGGCG AGATCCACAAGGGAAACCAGATCTTCCTGAGCCAGAAATTTGTGAAACTG CTGTACCACGCCAAGCGGATCTCCAACACCATCAATGAGAACCACCGGAA ATACGTGGAAAACCACAAGAAAGAGTTTGAGGAACTGTTCTACTACATCC TGGAGTTCAACGAGAACTATGTGGGAGCCAAGAAGAACGGCAAACTGCTG AACTCCGCCTTCCAGAGCTGGCAGAACCACAGCATCGACGAGCTGTGCAG CTCCTTCATCGGCCCTACCGGCAGCGAGCGGAAGGGACTGTTTGAGCTGA CCTCCAGAGGCTCTGCCGCCGACTTTGAGTTCCTGGGAGTGAAGATCCCC CGGTACAGAGACTACACCCCCTCTAGTCTGCTGAAGGACGCCACCCTGAT CCACCAGAGCGTGACCGGCCTGTACGAAACCCGGATCGACCTGGCTAAGC TGGGCGAGGGAAAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAG AAAAAGAAATAA
Example 5
Optimization of the Guide RNA for Streptococcus pyogenes Cas9 (Referred to as SpCas9)
[0212] Applicants mutated the tracrRNA and direct repeat sequences, or mutated the chimeric guide RNA to enhance the RNAs in cells.
[0213] The optimization is based on the observation that there were stretches of thymines (Ts) in the tracrRNA and guide RNA, which might lead to early transcription termination by the pol 3 promoter. Therefore Applicants generated the following optimized sequences. Optimized tracrRNA and corresponding optimized direct repeat are presented in pairs.
[0214] Optimized tracrRNA 1 (mutation underlined):
TABLE-US-00037 GGAACCATTCAtAACAGCATAGCAAGTTAtAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT
[0215] Optimized direct repeat 1 (mutation underlined):
TABLE-US-00038 GTTaTAGAGCTATGCTGTTaTGAATGGTCCCAAAAC
[0216] Optimized tracrRNA 2 (mutation underlined):
TABLE-US-00039 GGAACCATTCAAtACAGCATAGCAAGTTAAtATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT
[0217] Optimized direct repeat 2 (mutation underlined):
TABLE-US-00040 GTaTTAGAGCTATGCTGTaTTGAATGGTCCCAAAAC
[0218] Applicants also optimized the chimeric guideRNA for optimal activity in eukaryotic cells.
[0219] Original guide RNA:
TABLE-US-00041 NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAA TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTT
[0220] Optimized chimeric guide RNA sequence 1:
TABLE-US-00042 NNNNNNNNNNNNNNNNNNNNGTATTAGAGCTAGAAATAGCAAGTTAATA TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTT
Optimized chimeric guide RNA sequence 2:
TABLE-US-00043 NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTATGCTGTTTTGGAAACAAA ACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG TGGCACCGAGTCGGTGCTTTTTTT
[0221] Optimized chimeric guide RNA sequence 3:
TABLE-US-00044 NNNNNNNNNNNNNNNNNNNNGTATTAGAGCTATGCTGTATTGGAAACAA TACAGCATAGCAAGTTAATATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGCACCGAGTCGGTGCTTTTTTT
[0222] Applicants showed that optimized chimeric guide RNA works better as indicated in FIG. 9. The experiment was conducted by co-transfecting 293FT cells with Cas9 and a U6-guide RNA DNA cassette to express one of the four RNA forms shown above. The target of the guide RNA is the same target site in the human Emx 1 locus: "GTCACCTCCAATGACTAGGG"
Example 6
Optimization of Streptococcus thermophilus LMD-9 CRISPR1 Cas9 (referred to as St1Cas9)
[0223] Applicants designed guide chimeric RNAs as shown in FIG. 12.
[0224] The St1Cas9 guide RNAs can under go the same type of optimization as for SpCas9 guide RNAs, by breaking the stretches of poly thymines (Ts).
Example 7
Improvement of the Cas9 System for In Vivo Application
[0225] Applicants conducted a Metagenomic search for a Cas9 with small molecular weight. Most Cas9 homologs are fairly large. For example the SpCas9 is around 1368aa long, which is too large to be easily packaged into viral vectors for delivery. Some of the sequences may have been mis-annotated and therefore the exact frequency for each length may not necessarily be accurate. Nevertheless it provides a glimpse at distribution of Cas9 proteins and suggest that there are shorter Cas9 homologs.
[0226] Through computational analysis, Applicants found that in the bacterial strain Campylobacter, there are two Cas9 proteins with less than 1000 amino acids. The sequence for one Cas9 from Campylobacter jejuni is presented below. At this length, CjCas9 can be easily packaged into AAV, lentiviruses, Adenoviruses, and other viral vectors for robust delivery into primary cells and in vivo in animal models.
[0227] >Campylobacter jejuni Cas9 (CjCas9)
TABLE-US-00045 MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRL ARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLIS PYELRFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIK QNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFL KDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFT DEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLK NGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQD DLNEIAKLDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFK ALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTN PVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQN ENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDL QDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSA KWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFDRNLNDTRYIARLVLNY TKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKD RNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYK NKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQ SYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIY TMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKD MQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAK SIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK.
[0228] The putative tracrRNA element for this CjCas9 is:
TABLE-US-00046 TATAATCTCATAAGAAATTTAAAAAGGGACTAAAATAAAGAGTTTGCG GGACTCTGCGGGGTTACAATCCCCTAAAACCGCTTTTAAAATT
[0229] The Direct Repeat sequence is:
TABLE-US-00047 ATTTTACCATAAAGAAATTTAAAAAGGGACTAAAAC
[0230] The co-fold structure of the tracrRNA and direct repeat is provided in FIG. 6.
[0231] An example of a chimeric guide RNA for CjCas9 is:
TABLE-US-00048 NNNNNNNNNNNNNNNNNNNNGUUUUAGUCCCGAAAGGGACUAAAAUAA AGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU
[0232] Applicants have also optimized Cas9 guide RNA using in vitro methods. FIG. 18 shows data from the St1Cas9 chimeric guide RNA optimization in vitro.
[0233] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Example 8
Sa sgRNA Optimization
[0234] Applicants designed five sgRNA variants for SaCas9 for an optimal truncated architecture with highest cleavage efficiency. In addition, the native direct repeat:tracr duplex system was tested alongside sgRNAs. Guides with indicated lengths were co-transfected with SaCas9 and tested in HEK 293FT cells for activity. A total of 100 ng sgRNA U6-PCR amplicon (or 50 ng of direct repeat and 50 ng of tracrRNA) and 400 ng of SaCas9 plasmid were co-transfected into 200,000 Hepa1-6 mouse hepatocytes, and DNA was harvested 72-hours post-transfection for SURVEYOR analysis. The results are shown in FIG. 23.
REFERENCES
[0235] 1. Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636-646 (2010).
[0236] 2. Bogdanove, A. J. & Voytas, D. F. TAL effectors: customizable proteins for DNA targeting. Science 333, 1843-1846 (2011).
[0237] 3. Stoddard, B. L. Homing endonuclease structure and function. Q. Rev. Biophys. 38, 49-95 (2005).
[0238] 4. Bae, T. & Schneewind, O. Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid 55, 58-63 (2006).
[0239] 5. Sung, C. K., Li, H., Claverys, J. P. & Morrison, D. A. An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl. Environ. Microbiol. 67, 5190-5196 (2001).
[0240] 6. Sharan, S. K., Thomason, L. C., Kuznetsov, S. G. & Court, D. L. Recombineering: a homologous recombination-based method of genetic engineering. Nat. Protoc. 4, 206-223 (2009).
[0241] 7. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012).
[0242] 8. Deveau, H., Gameau, J. E. & Moineau, S. CRISPR-Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol. 64, 475-493 (2010).
[0243] 9. Horvath, P. & Barrangou, R. CRISPR-Cas, the immune system of bacteria and archaea. Science 327, 167-170 (2010).
[0244] 10. Terns, M. P. & Terns, R. M. CRISPR-based adaptive immune systems. Curr. Opin. Microbiol. 14, 321-327 (2011).
[0245] 11. van der Oost, J., Jore, M. M., Westra, E. R., Lundgren, M. & Brouns, S. J. CRISPR-based adaptive and heritable immunity in prokaryotes. Trends. Biochem. Sci. 34, 401-407 (2009).
[0246] 12. Brouns, S. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960-964 (2008).
[0247] 13. Carte, J., Wang, R., Li, H., Terns. R. M. & Terns, M. P. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 22, 3489-3496 (2008).
[0248] 14. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602-607 (2011).
[0249] 15. Hatoum-Aslan, A., Maniv, I. & Marraffini, L. A. Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc. Natl. Acad. Sci. U.S.A. 108, 21218-21222 (2011).
[0250] 16. Haurwitz, R. E., Jinek. M., Wiedenheft, B., Zhou, K. & Doudna, J. A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 1355-1358 (2010).
[0251] 17. Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 1390-1400 (2008).
[0252] 18. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. U.S.A. (2012).
[0253] 19. Makarova, K. S., Aravind, L., Wolf, Y. I. & Koonin, E. V. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol. Direct. 6, 38 (2011).
[0254] 20. Barrangou, R. RNA-mediated programmable DNA cleavage. Nat. Biotechnol. 30, 836-838 (2012).
[0255] 21. Brouns, S. J. Molecular biology. A Swiss army knife of immunity. Science 337, 808-809 (2012).
[0256] 22. Carroll, D. A CRISPR Approach to Gene Targeting. Mol. Ther. 20, 1658-1660 (2012).
[0257] 23. Bikard, D., Hatoum-Aslan, A., Mucida, D. & Marraffini, L. A. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Cell Host Microbe 12, 177-186 (2012).
[0258] 24. Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR-Cas system provides immunity in Escherichia coli. Nucleic Acids Res. (2011).
[0259] 25. Semenova, E. et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl. Acad. Sci. U.S.A. (2011).
[0260] 26. Wiedenheft, B. et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl. Acad. Sci. U.S.A. (2011).
[0261] 27. Zahner, D. & Hakenbeck, R. The Streptococcus pneumoniae beta-galactosidase is a surface protein. J. Bacteriol. 182, 5919-5921 (2000).
[0262] 28. Marraffini, L. A., Dedent, A. C. & Schneewind, O. Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiol. Mol. Biol. Rev. 70, 192-221 (2006).
[0263] 29. Motamedi, M. R., Szigety, S. K. & Rosenberg, S. M. Double-strand-break repair recombination in Escherichia coli: physical evidence for a DNA replication mechanism in vivo. Genes Dev. 13, 2889-2903 (1999).
[0264] 30. Hosaka, T. et al. The novel mutation K87E in ribosomal protein S12 enhances protein synthesis activity during the late growth phase in Escherichia coli. Mol. Genet. Genomics 271, 317-324 (2004).
[0265] 31. Costantino, N. & Court, D. L. Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants. Proc. Natl. Acad. Sci. U.S.A. 100, 15748-15753 (2003).
[0266] 32. Edgar, R. & Qimron. U. The Escherichia coli CRISPR system protects from lambda lysogenization, lysogens, and prophage induction. J. Bacteriol. 192, 6291-6294 (2010).
[0267] 33. Marraffini, L. A. & Sontheimer, E. J. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568-571 (2010).
[0268] 34. Fischer, S. et al. An archaeal immune system can detect multiple Protospacer Adjacent Motifs (PAMs) to target invader DNA. J. Biol. Chemn. 287, 33351-33363 (2012).
[0269] 35. Gudbergsdottir, S. et al. Dynamic properties of the Sulfolobus CRISPR-Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers. Mol. Microbiol. 79, 35-49 (2011).
[0270] 36. Wang, H. H. et al. Genome-scale promoter engineering by coselection MAGE. Nat Methods 9, 591-593 (2012).
[0271] 37. Cong, L. et al. Multiplex Genome Engineering Using CRISPR-Cas Systems. Science In press (2013).
[0272] 38. Mali, P. et al. RNA-Guided Human Genome Engineering via Cas9. Science In press (2013).
[0273] 39. Hoskins, J. et al. Genome of the bacterium Streptococcus pneumoniae strain R6. J. Bacteriol. 183, 5709-5717 (2001).
[0274] 40. Havarstein, L. S., Coomaraswamy, G. & Morrison, D. A. An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. U.S.A. 92, 11140-11144 (1995).
[0275] 41. Horinouchi, S. & Weisblum, B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150, 815-825 (1982).
[0276] 42. Horton, R. M. In Vitro Recombination and Mutagenesis of DNA: SOEing Together Tailor-Made Genes. Methods Mol. Biol. 15, 251-261 (1993).
[0277] 43. Podbielski, A., Spellerberg. B., Woischnik, M., Pohl, B. & Lutticken, R. Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS). Gene 177, 137-147 (1996).
[0278] 44. Husmann, L. K., Scott, J. R., Lindahl, G. & Stenberg, L. Expression of the Arp protein, a member of the M protein family, is not sufficient to inhibit phagocytosis of Streptococcus pyogenes. Infection and immunity 63, 345-348 (1995).
[0279] 45. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343-345 (2009).
[0280] 46. Tangri S, et al. ("Rationally engineered therapeutic proteins with reduced immunogenicity" J Immunol. 2005 Mar. 15; 174(6):3187-96.
[0281] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
Sequence CWU
1
1
264115DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 1aggacgaagt cctaa
1527PRTSimian virus 40 2Pro Lys Lys
Lys Arg Lys Val 1 5
316PRTUnknownsource/note="Description of Unknown Nucleoplasmin
bipartite NLS sequence" 3Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala
Lys Lys Lys Lys 1 5 10
15 49PRTUnknownsource/note="Description of Unknown C-myc NLS
sequence" 4Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5
511PRTUnknownsource/note="Description of Unknown C-myc NLS
sequence" 5Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro 1 5
10 638PRTHomo sapiens 6Asn Gln Ser Ser Asn Phe Gly
Pro Met Lys Gly Gly Asn Phe Gly Gly 1 5
10 15 Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln
Tyr Phe Ala Lys Pro 20 25
30 Arg Asn Gln Gly Gly Tyr 35
742PRTUnknownsource/note="Description of Unknown IBB domain from
importin-alpha sequence" 7Arg Met Arg Ile Glx Phe Lys Asn Lys Gly Lys Asp
Thr Ala Glu Leu 1 5 10
15 Arg Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys
20 25 30 Asp Glu Gln
Ile Leu Lys Arg Arg Asn Val 35 40
88PRTUnknownsource/note="Description of Unknown Myoma T protein
sequence" 8Val Ser Arg Lys Arg Pro Arg Pro 1 5
98PRTUnknownsource/note="Description of Unknown Myoma T protein
sequence" 9Pro Pro Lys Lys Ala Arg Glu Asp 1 5
108PRTHomo sapiens 10Pro Gln Pro Lys Lys Lys Pro Leu 1 5
1112PRTMus musculus 11Ser Ala Leu Ile Lys Lys Lys Lys Lys Met
Ala Pro 1 5 10 125PRTInfluenza
virus 12Asp Arg Leu Arg Arg 1 5 137PRTInfluenza virus
13Pro Lys Gln Lys Lys Arg Lys 1 5
1410PRTHepatitus delta virus 14Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu 1
5 10 1510PRTMus musculus 15Arg Glu Lys
Lys Lys Phe Leu Lys Arg Arg 1 5 10
1620PRTHomo sapiens 16Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val
Ala Lys Lys 1 5 10 15
Lys Ser Lys Lys 20 1717PRTHomo sapiens 17Arg Lys Cys Leu
Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys 1 5
10 15 Lys 1827DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 18nnnnnnnnnn nnnnnnnnnn nnagaaw
271919DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 19nnnnnnnnnn
nnnnagaaw
192027DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 20nnnnnnnnnn nnnnnnnnnn nnagaaw
272118DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 21nnnnnnnnnn nnnagaaw
1822137DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 22nnnnnnnnnn
nnnnnnnnnn gtttttgtac tctcaagatt tagaaataaa tcttgcagaa 60gctacaaaga
taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt 120tcgttattta
atttttt
13723123DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 23nnnnnnnnnn nnnnnnnnnn
gtttttgtac tctcagaaat gcagaagcta caaagataag 60gcttcatgcc gaaatcaaca
ccctgtcatt ttatggcagg gtgttttcgt tatttaattt 120ttt
12324110DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 24nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcagaaat gcagaagcta
caaagataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttttt
11025102DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 25nnnnnnnnnn
nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc 60cgttatcaac
ttgaaaaagt ggcaccgagt cggtgctttt tt
1022688DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 26nnnnnnnnnn nnnnnnnnnn
gttttagagc tagaaatagc aagttaaaat aaggctagtc 60cgttatcaac ttgaaaaagt
gttttttt 882776DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 27nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat
aaggctagtc 60cgttatcatt tttttt
762812RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 28guuuuagagc ua
122933DNAHomo sapiens
29ggacatcgat gtcacctcca atgactaggg tgg
333033DNAHomo sapiens 30cattggaggt gacatcgatg tcctccccat tgg
333133DNAHomo sapiens 31ggaagggcct gagtccgagc
agaagaagaa ggg 333233DNAHomo sapiens
32ggtggcgaga ggggccgaga ttgggtgttc agg
333333DNAHomo sapiens 33atgcaggagg gtggcgagag gggccgagat tgg
333421DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic primer" 34aaaaccaccc ttctctctgg c
213521DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 35ggagattgga gacacggaga g
213620DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 36ctggaaagcc aatgcctgac
203720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 37ggcagcaaac tccttgtcct
203812DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 38gttttagagc ta
1239335DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 39gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
tgttagagag 60ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
gtgacgtaga 120aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
ggactatcat 180atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
gtggaaagga 240cgaaacaccg gaaccattca aaacagcata gcaagttaaa ataaggctag
tccgttatca 300acttgaaaaa gtggcaccga gtcggtgctt ttttt
33540423DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 40gagggcctat
ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60ataattggaa
ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120aagtaataat
ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180atgcttaccg
taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240cgaaacaccg
gtagtattaa gtattgtttt atggctgata aatttctttg aatttctcct 300tgattatttg
ttataaaagt tataaaataa tcttgttgga accattcaaa acagcatagc 360aagttaaaat
aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 420ttt
42341339DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 41gagggcctat ttcccatgat
tccttcatat ttgcatatac gatacaaggc tgttagagag 60ataattggaa ttaatttgac
tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120aagtaataat ttcttgggta
gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180atgcttaccg taacttgaaa
gtatttcgat ttcttggctt tatatatctt gtggaaagga 240cgaaacaccg ggttttagag
ctatgctgtt ttgaatggtc ccaaaacggg tcttcgagaa 300gacgttttag agctatgctg
ttttgaatgg tcccaaaac 33942309DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 42gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
tgttagagag 60ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
gtgacgtaga 120aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
ggactatcat 180atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
gtggaaagga 240cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag
ttaaaataag 300gctagtccg
309431648PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 43Met Asp Tyr Lys Asp His
Asp Gly Asp Tyr Lys Asp His Asp Ile Asp 1 5
10 15 Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys
Lys Lys Arg Lys Val 20 25
30 Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly
Leu 35 40 45 Asp
Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr 50
55 60 Lys Val Pro Ser Lys Lys
Phe Lys Val Leu Gly Asn Thr Asp Arg His 65 70
75 80 Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu
Phe Asp Ser Gly Glu 85 90
95 Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr
100 105 110 Arg Arg
Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu 115
120 125 Met Ala Lys Val Asp Asp Ser
Phe Phe His Arg Leu Glu Glu Ser Phe 130 135
140 Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro
Ile Phe Gly Asn 145 150 155
160 Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His
165 170 175 Leu Arg Lys
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu 180
185 190 Ile Tyr Leu Ala Leu Ala His Met
Ile Lys Phe Arg Gly His Phe Leu 195 200
205 Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp
Lys Leu Phe 210 215 220
Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile 225
230 235 240 Asn Ala Ser Gly
Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser 245
250 255 Lys Ser Arg Arg Leu Glu Asn Leu Ile
Ala Gln Leu Pro Gly Glu Lys 260 265
270 Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly
Leu Thr 275 280 285
Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln 290
295 300 Leu Ser Lys Asp Thr
Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln 305 310
315 320 Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu
Ala Ala Lys Asn Leu Ser 325 330
335 Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
Thr 340 345 350 Lys
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His 355
360 365 Gln Asp Leu Thr Leu Leu
Lys Ala Leu Val Arg Gln Gln Leu Pro Glu 370 375
380 Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys
Asn Gly Tyr Ala Gly 385 390 395
400 Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys
405 410 415 Pro Ile
Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu 420
425 430 Asn Arg Glu Asp Leu Leu Arg
Lys Gln Arg Thr Phe Asp Asn Gly Ser 435 440
445 Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala
Ile Leu Arg Arg 450 455 460
Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu 465
470 475 480 Lys Ile Leu
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg 485
490 495 Gly Asn Ser Arg Phe Ala Trp Met
Thr Arg Lys Ser Glu Glu Thr Ile 500 505
510 Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala
Ser Ala Gln 515 520 525
Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu 530
535 540 Lys Val Leu Pro
Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr 545 550
555 560 Asn Glu Leu Thr Lys Val Lys Tyr Val
Thr Glu Gly Met Arg Lys Pro 565 570
575 Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu
Leu Phe 580 585 590
Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe
595 600 605 Lys Lys Ile Glu
Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp 610
615 620 Arg Phe Asn Ala Ser Leu Gly Thr
Tyr His Asp Leu Leu Lys Ile Ile 625 630
635 640 Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu
Asp Ile Leu Glu 645 650
655 Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu
660 665 670 Glu Arg Leu
Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys 675
680 685 Gln Leu Lys Arg Arg Arg Tyr Thr
Gly Trp Gly Arg Leu Ser Arg Lys 690 695
700 Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr
Ile Leu Asp 705 710 715
720 Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
725 730 735 His Asp Asp Ser
Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val 740
745 750 Ser Gly Gln Gly Asp Ser Leu His Glu
His Ile Ala Asn Leu Ala Gly 755 760
765 Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val
Val Asp 770 775 780
Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile 785
790 795 800 Glu Met Ala Arg Glu
Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 805
810 815 Arg Glu Arg Met Lys Arg Ile Glu Glu Gly
Ile Lys Glu Leu Gly Ser 820 825
830 Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn
Glu 835 840 845 Lys
Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp 850
855 860 Gln Glu Leu Asp Ile Asn
Arg Leu Ser Asp Tyr Asp Val Asp His Ile 865 870
875 880 Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
Asp Asn Lys Val Leu 885 890
895 Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu
900 905 910 Glu Val
Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala 915
920 925 Lys Leu Ile Thr Gln Arg Lys
Phe Asp Asn Leu Thr Lys Ala Glu Arg 930 935
940 Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile
Lys Arg Gln Leu 945 950 955
960 Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
965 970 975 Arg Met Asn
Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val 980
985 990 Lys Val Ile Thr Leu Lys Ser Lys
Leu Val Ser Asp Phe Arg Lys Asp 995 1000
1005 Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn
Tyr His His Ala 1010 1015 1020
His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys
1025 1030 1035 Lys Tyr Pro
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys 1040
1045 1050 Val Tyr Asp Val Arg Lys Met Ile
Ala Lys Ser Glu Gln Glu Ile 1055 1060
1065 Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile
Met Asn 1070 1075 1080
Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1085
1090 1095 Arg Pro Leu Ile Glu
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp 1100 1105
1110 Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
Lys Val Leu Ser Met 1115 1120 1125
Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1130 1135 1140 Phe Ser
Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu 1145
1150 1155 Ile Ala Arg Lys Lys Asp Trp
Asp Pro Lys Lys Tyr Gly Gly Phe 1160 1165
1170 Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val
Ala Lys Val 1175 1180 1185
Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu 1190
1195 1200 Gly Ile Thr Ile Met
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile 1205 1210
1215 Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
Val Lys Lys Asp Leu 1220 1225 1230
Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly
1235 1240 1245 Arg Lys
Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn 1250
1255 1260 Glu Leu Ala Leu Pro Ser Lys
Tyr Val Asn Phe Leu Tyr Leu Ala 1265 1270
1275 Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp
Asn Glu Gln 1280 1285 1290
Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile 1295
1300 1305 Ile Glu Gln Ile Ser
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp 1310 1315
1320 Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
Asn Lys His Arg Asp 1325 1330 1335
Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr
1340 1345 1350 Leu Thr
Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr 1355
1360 1365 Thr Ile Asp Arg Lys Arg Tyr
Thr Ser Thr Lys Glu Val Leu Asp 1370 1375
1380 Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr
Glu Thr Arg 1385 1390 1395
Ile Asp Leu Ser Gln Leu Gly Gly Asp Ala Ala Ala Val Ser Lys 1400
1405 1410 Gly Glu Glu Leu Phe
Thr Gly Val Val Pro Ile Leu Val Glu Leu 1415 1420
1425 Asp Gly Asp Val Asn Gly His Lys Phe Ser
Val Ser Gly Glu Gly 1430 1435 1440
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
1445 1450 1455 Thr Thr
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 1460
1465 1470 Leu Thr Tyr Gly Val Gln Cys
Phe Ser Arg Tyr Pro Asp His Met 1475 1480
1485 Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu
Gly Tyr Val 1490 1495 1500
Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 1505
1510 1515 Arg Ala Glu Val Lys
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 1520 1525
1530 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp
Gly Asn Ile Leu Gly 1535 1540 1545
His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met
1550 1555 1560 Ala Asp
Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg 1565
1570 1575 His Asn Ile Glu Asp Gly Ser
Val Gln Leu Ala Asp His Tyr Gln 1580 1585
1590 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu
Pro Asp Asn 1595 1600 1605
His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 1610
1615 1620 Lys Arg Asp His Met
Val Leu Leu Glu Phe Val Thr Ala Ala Gly 1625 1630
1635 Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
1640 1645 441625PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 44Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn
Ser Val 1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30 Lys Val Leu Gly Asn
Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35
40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu
Thr Ala Glu Ala Thr Arg Leu 50 55
60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn
Arg Ile Cys 65 70 75
80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95 Phe Phe His Arg
Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100
105 110 His Glu Arg His Pro Ile Phe Gly Asn
Ile Val Asp Glu Val Ala Tyr 115 120
125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu
Val Asp 130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145
150 155 160 Met Ile Lys Phe Arg
Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165
170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile
Gln Leu Val Gln Thr Tyr 180 185
190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp
Ala 195 200 205 Lys
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210
215 220 Leu Ile Ala Gln Leu Pro
Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230
235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn
Phe Lys Ser Asn Phe 245 250
255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270 Asp Asp
Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275
280 285 Leu Phe Leu Ala Ala Lys Asn
Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295
300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
Leu Ser Ala Ser 305 310 315
320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335 Ala Leu Val
Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340
345 350 Asp Gln Ser Lys Asn Gly Tyr Ala
Gly Tyr Ile Asp Gly Gly Ala Ser 355 360
365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu
Lys Met Asp 370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385
390 395 400 Lys Gln Arg Thr
Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405
410 415 Gly Glu Leu His Ala Ile Leu Arg Arg
Gln Glu Asp Phe Tyr Pro Phe 420 425
430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe
Arg Ile 435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450
455 460 Met Thr Arg Lys Ser
Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470
475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser
Phe Ile Glu Arg Met Thr 485 490
495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His
Ser 500 505 510 Leu
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515
520 525 Tyr Val Thr Glu Gly Met
Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535
540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr
Asn Arg Lys Val Thr 545 550 555
560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575 Ser Val
Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580
585 590 Thr Tyr His Asp Leu Leu Lys
Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600
605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
Leu Thr Leu Thr 610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625
630 635 640 His Leu Phe
Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645
650 655 Thr Gly Trp Gly Arg Leu Ser Arg
Lys Leu Ile Asn Gly Ile Arg Asp 660 665
670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser
Asp Gly Phe 675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690
695 700 Lys Glu Asp Ile
Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710
715 720 His Glu His Ile Ala Asn Leu Ala Gly
Ser Pro Ala Ile Lys Lys Gly 725 730
735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val
Met Gly 740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765 Thr Thr Gln Lys
Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770
775 780 Glu Glu Gly Ile Lys Glu Leu Gly
Ser Gln Ile Leu Lys Glu His Pro 785 790
795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr
Leu Tyr Tyr Leu 805 810
815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830 Leu Ser Asp
Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835
840 845 Asp Asp Ser Ile Asp Asn Lys Val
Leu Thr Arg Ser Asp Lys Asn Arg 850 855
860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys
Lys Met Lys 865 870 875
880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895 Phe Asp Asn Leu
Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900
905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu
Val Glu Thr Arg Gln Ile Thr 915 920
925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
Tyr Asp 930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945
950 955 960 Lys Leu Val Ser Asp
Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965
970 975 Glu Ile Asn Asn Tyr His His Ala His Asp
Ala Tyr Leu Asn Ala Val 980 985
990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
Glu Phe 995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010
1015 1020 Lys Ser Glu Gln Glu
Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030
1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr
Glu Ile Thr Leu Ala 1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065 Thr Gly
Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070
1075 1080 Arg Lys Val Leu Ser Met Pro
Gln Val Asn Ile Val Lys Lys Thr 1085 1090
1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile
Leu Pro Lys 1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115
1120 1125 Lys Lys Tyr Gly Gly
Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135
1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys
Ser Lys Lys Leu Lys 1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170 Phe Glu
Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175
1180 1185 Glu Val Lys Lys Asp Leu Ile
Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195
1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala
Ser Ala Gly 1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220
1225 1230 Asn Phe Leu Tyr Leu
Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240
1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe
Val Glu Gln His Lys 1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275 Arg Val
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280
1285 1290 Tyr Asn Lys His Arg Asp Lys
Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300
1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala
Pro Ala Ala 1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325
1330 1335 Thr Lys Glu Val Leu
Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345
1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser
Gln Leu Gly Gly Asp 1355 1360 1365
Ala Ala Ala Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val
1370 1375 1380 Pro Ile
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 1385
1390 1395 Ser Val Ser Gly Glu Gly Glu
Gly Asp Ala Thr Tyr Gly Lys Leu 1400 1405
1410 Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro
Val Pro Trp 1415 1420 1425
Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser 1430
1435 1440 Arg Tyr Pro Asp His
Met Lys Gln His Asp Phe Phe Lys Ser Ala 1445 1450
1455 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr
Ile Phe Phe Lys Asp 1460 1465 1470
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
1475 1480 1485 Thr Leu
Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 1490
1495 1500 Asp Gly Asn Ile Leu Gly His
Lys Leu Glu Tyr Asn Tyr Asn Ser 1505 1510
1515 His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
Gly Ile Lys 1520 1525 1530
Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln 1535
1540 1545 Leu Ala Asp His Tyr
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 1550 1555
1560 Val Leu Leu Pro Asp Asn His Tyr Leu Ser
Thr Gln Ser Ala Leu 1565 1570 1575
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
1580 1585 1590 Phe Val
Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr 1595
1600 1605 Lys Lys Arg Pro Ala Ala Thr
Lys Lys Ala Gly Gln Ala Lys Lys 1610 1615
1620 Lys Lys 1625 451664PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 45Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp
Ile Asp 1 5 10 15
Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val
20 25 30 Gly Ile His Gly Val
Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu 35
40 45 Asp Ile Gly Thr Asn Ser Val Gly Trp
Ala Val Ile Thr Asp Glu Tyr 50 55
60 Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr
Asp Arg His 65 70 75
80 Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu
85 90 95 Thr Ala Glu Ala
Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr 100
105 110 Arg Arg Lys Asn Arg Ile Cys Tyr Leu
Gln Glu Ile Phe Ser Asn Glu 115 120
125 Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu
Ser Phe 130 135 140
Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn 145
150 155 160 Ile Val Asp Glu Val
Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His 165
170 175 Leu Arg Lys Lys Leu Val Asp Ser Thr Asp
Lys Ala Asp Leu Arg Leu 180 185
190 Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
Leu 195 200 205 Ile
Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe 210
215 220 Ile Gln Leu Val Gln Thr
Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile 225 230
235 240 Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu
Ser Ala Arg Leu Ser 245 250
255 Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys
260 265 270 Lys Asn
Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr 275
280 285 Pro Asn Phe Lys Ser Asn Phe
Asp Leu Ala Glu Asp Ala Lys Leu Gln 290 295
300 Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn
Leu Leu Ala Gln 305 310 315
320 Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser
325 330 335 Asp Ala Ile
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr 340
345 350 Lys Ala Pro Leu Ser Ala Ser Met
Ile Lys Arg Tyr Asp Glu His His 355 360
365 Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln
Leu Pro Glu 370 375 380
Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly 385
390 395 400 Tyr Ile Asp Gly
Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys 405
410 415 Pro Ile Leu Glu Lys Met Asp Gly Thr
Glu Glu Leu Leu Val Lys Leu 420 425
430 Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn
Gly Ser 435 440 445
Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg 450
455 460 Gln Glu Asp Phe Tyr
Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu 465 470
475 480 Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr
Val Gly Pro Leu Ala Arg 485 490
495 Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
Ile 500 505 510 Thr
Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln 515
520 525 Ser Phe Ile Glu Arg Met
Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu 530 535
540 Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu
Tyr Phe Thr Val Tyr 545 550 555
560 Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro
565 570 575 Ala Phe
Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe 580
585 590 Lys Thr Asn Arg Lys Val Thr
Val Lys Gln Leu Lys Glu Asp Tyr Phe 595 600
605 Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser
Gly Val Glu Asp 610 615 620
Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile 625
630 635 640 Lys Asp Lys
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu 645
650 655 Asp Ile Val Leu Thr Leu Thr Leu
Phe Glu Asp Arg Glu Met Ile Glu 660 665
670 Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys
Val Met Lys 675 680 685
Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys 690
695 700 Leu Ile Asn Gly
Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp 705 710
715 720 Phe Leu Lys Ser Asp Gly Phe Ala Asn
Arg Asn Phe Met Gln Leu Ile 725 730
735 His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala
Gln Val 740 745 750
Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly
755 760 765 Ser Pro Ala Ile
Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp 770
775 780 Glu Leu Val Lys Val Met Gly Arg
His Lys Pro Glu Asn Ile Val Ile 785 790
795 800 Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly
Gln Lys Asn Ser 805 810
815 Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser
820 825 830 Gln Ile Leu
Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu 835
840 845 Lys Leu Tyr Leu Tyr Tyr Leu Gln
Asn Gly Arg Asp Met Tyr Val Asp 850 855
860 Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val
Asp His Ile 865 870 875
880 Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu
885 890 895 Thr Arg Ser Asp
Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu 900
905 910 Glu Val Val Lys Lys Met Lys Asn Tyr
Trp Arg Gln Leu Leu Asn Ala 915 920
925 Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala
Glu Arg 930 935 940
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu 945
950 955 960 Val Glu Thr Arg Gln
Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser 965
970 975 Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp
Lys Leu Ile Arg Glu Val 980 985
990 Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg
Lys Asp 995 1000 1005
Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala 1010
1015 1020 His Asp Ala Tyr Leu
Asn Ala Val Val Gly Thr Ala Leu Ile Lys 1025 1030
1035 Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
Tyr Gly Asp Tyr Lys 1040 1045 1050
Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile
1055 1060 1065 Gly Lys
Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn 1070
1075 1080 Phe Phe Lys Thr Glu Ile Thr
Leu Ala Asn Gly Glu Ile Arg Lys 1085 1090
1095 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu
Ile Val Trp 1100 1105 1110
Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met 1115
1120 1125 Pro Gln Val Asn Ile
Val Lys Lys Thr Glu Val Gln Thr Gly Gly 1130 1135
1140 Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
Asn Ser Asp Lys Leu 1145 1150 1155
Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe
1160 1165 1170 Asp Ser
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val 1175
1180 1185 Glu Lys Gly Lys Ser Lys Lys
Leu Lys Ser Val Lys Glu Leu Leu 1190 1195
1200 Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys
Asn Pro Ile 1205 1210 1215
Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu 1220
1225 1230 Ile Ile Lys Leu Pro
Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly 1235 1240
1245 Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
Leu Gln Lys Gly Asn 1250 1255 1260
Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala
1265 1270 1275 Ser His
Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln 1280
1285 1290 Lys Gln Leu Phe Val Glu Gln
His Lys His Tyr Leu Asp Glu Ile 1295 1300
1305 Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile
Leu Ala Asp 1310 1315 1320
Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1325
1330 1335 Lys Pro Ile Arg Glu
Gln Ala Glu Asn Ile Ile His Leu Phe Thr 1340 1345
1350 Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
Lys Tyr Phe Asp Thr 1355 1360 1365
Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp
1370 1375 1380 Ala Thr
Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg 1385
1390 1395 Ile Asp Leu Ser Gln Leu Gly
Gly Asp Ala Ala Ala Val Ser Lys 1400 1405
1410 Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
Val Glu Leu 1415 1420 1425
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly 1430
1435 1440 Glu Gly Asp Ala Thr
Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 1445 1450
1455 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
Thr Leu Val Thr Thr 1460 1465 1470
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met
1475 1480 1485 Lys Gln
His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val 1490
1495 1500 Gln Glu Arg Thr Ile Phe Phe
Lys Asp Asp Gly Asn Tyr Lys Thr 1505 1510
1515 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val
Asn Arg Ile 1520 1525 1530
Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly 1535
1540 1545 His Lys Leu Glu Tyr
Asn Tyr Asn Ser His Asn Val Tyr Ile Met 1550 1555
1560 Ala Asp Lys Gln Lys Asn Gly Ile Lys Val
Asn Phe Lys Ile Arg 1565 1570 1575
His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln
1580 1585 1590 Gln Asn
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn 1595
1600 1605 His Tyr Leu Ser Thr Gln Ser
Ala Leu Ser Lys Asp Pro Asn Glu 1610 1615
1620 Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr
Ala Ala Gly 1625 1630 1635
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Lys Arg Pro Ala Ala 1640
1645 1650 Thr Lys Lys Ala Gly
Gln Ala Lys Lys Lys Lys 1655 1660
461423PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 46Met Asp Tyr Lys Asp His Asp Gly
Asp Tyr Lys Asp His Asp Ile Asp 1 5 10
15 Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys
Arg Lys Val 20 25 30
Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu
35 40 45 Asp Ile Gly Thr
Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr 50
55 60 Lys Val Pro Ser Lys Lys Phe Lys
Val Leu Gly Asn Thr Asp Arg His 65 70
75 80 Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe
Asp Ser Gly Glu 85 90
95 Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr
100 105 110 Arg Arg Lys
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu 115
120 125 Met Ala Lys Val Asp Asp Ser Phe
Phe His Arg Leu Glu Glu Ser Phe 130 135
140 Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile
Phe Gly Asn 145 150 155
160 Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His
165 170 175 Leu Arg Lys Lys
Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu 180
185 190 Ile Tyr Leu Ala Leu Ala His Met Ile
Lys Phe Arg Gly His Phe Leu 195 200
205 Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys
Leu Phe 210 215 220
Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile 225
230 235 240 Asn Ala Ser Gly Val
Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser 245
250 255 Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala
Gln Leu Pro Gly Glu Lys 260 265
270 Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
Thr 275 280 285 Pro
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln 290
295 300 Leu Ser Lys Asp Thr Tyr
Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln 305 310
315 320 Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
Ala Lys Asn Leu Ser 325 330
335 Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr
340 345 350 Lys Ala
Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His 355
360 365 Gln Asp Leu Thr Leu Leu Lys
Ala Leu Val Arg Gln Gln Leu Pro Glu 370 375
380 Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn
Gly Tyr Ala Gly 385 390 395
400 Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys
405 410 415 Pro Ile Leu
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu 420
425 430 Asn Arg Glu Asp Leu Leu Arg Lys
Gln Arg Thr Phe Asp Asn Gly Ser 435 440
445 Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile
Leu Arg Arg 450 455 460
Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu 465
470 475 480 Lys Ile Leu Thr
Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg 485
490 495 Gly Asn Ser Arg Phe Ala Trp Met Thr
Arg Lys Ser Glu Glu Thr Ile 500 505
510 Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser
Ala Gln 515 520 525
Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu 530
535 540 Lys Val Leu Pro Lys
His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr 545 550
555 560 Asn Glu Leu Thr Lys Val Lys Tyr Val Thr
Glu Gly Met Arg Lys Pro 565 570
575 Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
Phe 580 585 590 Lys
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe 595
600 605 Lys Lys Ile Glu Cys Phe
Asp Ser Val Glu Ile Ser Gly Val Glu Asp 610 615
620 Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
Leu Leu Lys Ile Ile 625 630 635
640 Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu
645 650 655 Asp Ile
Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu 660
665 670 Glu Arg Leu Lys Thr Tyr Ala
His Leu Phe Asp Asp Lys Val Met Lys 675 680
685 Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg
Leu Ser Arg Lys 690 695 700
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp 705
710 715 720 Phe Leu Lys
Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile 725
730 735 His Asp Asp Ser Leu Thr Phe Lys
Glu Asp Ile Gln Lys Ala Gln Val 740 745
750 Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn
Leu Ala Gly 755 760 765
Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp 770
775 780 Glu Leu Val Lys
Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile 785 790
795 800 Glu Met Ala Arg Glu Asn Gln Thr Thr
Gln Lys Gly Gln Lys Asn Ser 805 810
815 Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu
Gly Ser 820 825 830
Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
835 840 845 Lys Leu Tyr Leu
Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp 850
855 860 Gln Glu Leu Asp Ile Asn Arg Leu
Ser Asp Tyr Asp Val Asp His Ile 865 870
875 880 Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp
Asn Lys Val Leu 885 890
895 Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu
900 905 910 Glu Val Val
Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala 915
920 925 Lys Leu Ile Thr Gln Arg Lys Phe
Asp Asn Leu Thr Lys Ala Glu Arg 930 935
940 Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys
Arg Gln Leu 945 950 955
960 Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
965 970 975 Arg Met Asn Thr
Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val 980
985 990 Lys Val Ile Thr Leu Lys Ser Lys
Leu Val Ser Asp Phe Arg Lys Asp 995 1000
1005 Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn
Tyr His His Ala 1010 1015 1020
His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys
1025 1030 1035 Lys Tyr Pro
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys 1040
1045 1050 Val Tyr Asp Val Arg Lys Met Ile
Ala Lys Ser Glu Gln Glu Ile 1055 1060
1065 Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile
Met Asn 1070 1075 1080
Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1085
1090 1095 Arg Pro Leu Ile Glu
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp 1100 1105
1110 Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
Lys Val Leu Ser Met 1115 1120 1125
Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1130 1135 1140 Phe Ser
Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu 1145
1150 1155 Ile Ala Arg Lys Lys Asp Trp
Asp Pro Lys Lys Tyr Gly Gly Phe 1160 1165
1170 Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val
Ala Lys Val 1175 1180 1185
Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu 1190
1195 1200 Gly Ile Thr Ile Met
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile 1205 1210
1215 Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
Val Lys Lys Asp Leu 1220 1225 1230
Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly
1235 1240 1245 Arg Lys
Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn 1250
1255 1260 Glu Leu Ala Leu Pro Ser Lys
Tyr Val Asn Phe Leu Tyr Leu Ala 1265 1270
1275 Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp
Asn Glu Gln 1280 1285 1290
Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile 1295
1300 1305 Ile Glu Gln Ile Ser
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp 1310 1315
1320 Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
Asn Lys His Arg Asp 1325 1330 1335
Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr
1340 1345 1350 Leu Thr
Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr 1355
1360 1365 Thr Ile Asp Arg Lys Arg Tyr
Thr Ser Thr Lys Glu Val Leu Asp 1370 1375
1380 Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr
Glu Thr Arg 1385 1390 1395
Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr 1400
1405 1410 Lys Lys Ala Gly Gln
Ala Lys Lys Lys Lys 1415 1420
47483PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 47Met Phe Leu Phe Leu Ser Leu Thr
Ser Phe Leu Ser Ser Ser Arg Thr 1 5 10
15 Leu Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile
Lys Glu Phe 20 25 30
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
35 40 45 Glu Ile Glu Gly
Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr 50
55 60 Ala Lys Leu Lys Val Thr Lys Gly
Gly Pro Leu Pro Phe Ala Trp Asp 65 70
75 80 Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala
Tyr Val Lys His 85 90
95 Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
100 105 110 Lys Trp Glu
Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val 115
120 125 Thr Gln Asp Ser Ser Leu Gln Asp
Gly Glu Phe Ile Tyr Lys Val Lys 130 135
140 Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met
Gln Lys Lys 145 150 155
160 Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
165 170 175 Ala Leu Lys Gly
Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly 180
185 190 His Tyr Asp Ala Glu Val Lys Thr Thr
Tyr Lys Ala Lys Lys Pro Val 195 200
205 Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile
Thr Ser 210 215 220
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly 225
230 235 240 Arg His Ser Thr Gly
Gly Met Asp Glu Leu Tyr Lys Gly Ser Lys Gln 245
250 255 Leu Glu Glu Leu Leu Ser Thr Ser Phe Asp
Ile Gln Phe Asn Asp Leu 260 265
270 Thr Leu Leu Glu Thr Ala Phe Thr His Thr Ser Tyr Ala Asn Glu
His 275 280 285 Arg
Leu Leu Asn Val Ser His Asn Glu Arg Leu Glu Phe Leu Gly Asp 290
295 300 Ala Val Leu Gln Leu Ile
Ile Ser Glu Tyr Leu Phe Ala Lys Tyr Pro 305 310
315 320 Lys Lys Thr Glu Gly Asp Met Ser Lys Leu Arg
Ser Met Ile Val Arg 325 330
335 Glu Glu Ser Leu Ala Gly Phe Ser Arg Phe Cys Ser Phe Asp Ala Tyr
340 345 350 Ile Lys
Leu Gly Lys Gly Glu Glu Lys Ser Gly Gly Arg Arg Arg Asp 355
360 365 Thr Ile Leu Gly Asp Leu Phe
Glu Ala Phe Leu Gly Ala Leu Leu Leu 370 375
380 Asp Lys Gly Ile Asp Ala Val Arg Arg Phe Leu Lys
Gln Val Met Ile 385 390 395
400 Pro Gln Val Glu Lys Gly Asn Phe Glu Arg Val Lys Asp Tyr Lys Thr
405 410 415 Cys Leu Gln
Glu Phe Leu Gln Thr Lys Gly Asp Val Ala Ile Asp Tyr 420
425 430 Gln Val Ile Ser Glu Lys Gly Pro
Ala His Ala Lys Gln Phe Glu Val 435 440
445 Ser Ile Val Val Asn Gly Ala Val Leu Ser Lys Gly Leu
Gly Lys Ser 450 455 460
Lys Lys Leu Ala Glu Gln Asp Ala Ala Lys Asn Ala Leu Ala Gln Leu 465
470 475 480 Ser Glu Val
48483PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 48Met Lys Gln Leu Glu Glu Leu Leu
Ser Thr Ser Phe Asp Ile Gln Phe 1 5 10
15 Asn Asp Leu Thr Leu Leu Glu Thr Ala Phe Thr His Thr
Ser Tyr Ala 20 25 30
Asn Glu His Arg Leu Leu Asn Val Ser His Asn Glu Arg Leu Glu Phe
35 40 45 Leu Gly Asp Ala
Val Leu Gln Leu Ile Ile Ser Glu Tyr Leu Phe Ala 50
55 60 Lys Tyr Pro Lys Lys Thr Glu Gly
Asp Met Ser Lys Leu Arg Ser Met 65 70
75 80 Ile Val Arg Glu Glu Ser Leu Ala Gly Phe Ser Arg
Phe Cys Ser Phe 85 90
95 Asp Ala Tyr Ile Lys Leu Gly Lys Gly Glu Glu Lys Ser Gly Gly Arg
100 105 110 Arg Arg Asp
Thr Ile Leu Gly Asp Leu Phe Glu Ala Phe Leu Gly Ala 115
120 125 Leu Leu Leu Asp Lys Gly Ile Asp
Ala Val Arg Arg Phe Leu Lys Gln 130 135
140 Val Met Ile Pro Gln Val Glu Lys Gly Asn Phe Glu Arg
Val Lys Asp 145 150 155
160 Tyr Lys Thr Cys Leu Gln Glu Phe Leu Gln Thr Lys Gly Asp Val Ala
165 170 175 Ile Asp Tyr Gln
Val Ile Ser Glu Lys Gly Pro Ala His Ala Lys Gln 180
185 190 Phe Glu Val Ser Ile Val Val Asn Gly
Ala Val Leu Ser Lys Gly Leu 195 200
205 Gly Lys Ser Lys Lys Leu Ala Glu Gln Asp Ala Ala Lys Asn
Ala Leu 210 215 220
Ala Gln Leu Ser Glu Val Gly Ser Val Ser Lys Gly Glu Glu Asp Asn 225
230 235 240 Met Ala Ile Ile Lys
Glu Phe Met Arg Phe Lys Val His Met Glu Gly 245
250 255 Ser Val Asn Gly His Glu Phe Glu Ile Glu
Gly Glu Gly Glu Gly Arg 260 265
270 Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly
Gly 275 280 285 Pro
Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly 290
295 300 Ser Lys Ala Tyr Val Lys
His Pro Ala Asp Ile Pro Asp Tyr Leu Lys 305 310
315 320 Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg
Val Met Asn Phe Glu 325 330
335 Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly
340 345 350 Glu Phe
Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp 355
360 365 Gly Pro Val Met Gln Lys Lys
Thr Met Gly Trp Glu Ala Ser Ser Glu 370 375
380 Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu
Ile Lys Gln Arg 385 390 395
400 Leu Lys Leu Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr
405 410 415 Tyr Lys Ala
Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn 420
425 430 Ile Lys Leu Asp Ile Thr Ser His
Asn Glu Asp Tyr Thr Ile Val Glu 435 440
445 Gln Tyr Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly
Met Asp Glu 450 455 460
Leu Tyr Lys Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys 465
470 475 480 Lys Lys Lys
491423PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 49Met Asp Tyr Lys Asp His Asp Gly
Asp Tyr Lys Asp His Asp Ile Asp 1 5 10
15 Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys
Arg Lys Val 20 25 30
Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu
35 40 45 Ala Ile Gly Thr
Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr 50
55 60 Lys Val Pro Ser Lys Lys Phe Lys
Val Leu Gly Asn Thr Asp Arg His 65 70
75 80 Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe
Asp Ser Gly Glu 85 90
95 Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr
100 105 110 Arg Arg Lys
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu 115
120 125 Met Ala Lys Val Asp Asp Ser Phe
Phe His Arg Leu Glu Glu Ser Phe 130 135
140 Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile
Phe Gly Asn 145 150 155
160 Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His
165 170 175 Leu Arg Lys Lys
Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu 180
185 190 Ile Tyr Leu Ala Leu Ala His Met Ile
Lys Phe Arg Gly His Phe Leu 195 200
205 Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys
Leu Phe 210 215 220
Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile 225
230 235 240 Asn Ala Ser Gly Val
Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser 245
250 255 Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala
Gln Leu Pro Gly Glu Lys 260 265
270 Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
Thr 275 280 285 Pro
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln 290
295 300 Leu Ser Lys Asp Thr Tyr
Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln 305 310
315 320 Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala
Ala Lys Asn Leu Ser 325 330
335 Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr
340 345 350 Lys Ala
Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His 355
360 365 Gln Asp Leu Thr Leu Leu Lys
Ala Leu Val Arg Gln Gln Leu Pro Glu 370 375
380 Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn
Gly Tyr Ala Gly 385 390 395
400 Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys
405 410 415 Pro Ile Leu
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu 420
425 430 Asn Arg Glu Asp Leu Leu Arg Lys
Gln Arg Thr Phe Asp Asn Gly Ser 435 440
445 Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile
Leu Arg Arg 450 455 460
Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu 465
470 475 480 Lys Ile Leu Thr
Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg 485
490 495 Gly Asn Ser Arg Phe Ala Trp Met Thr
Arg Lys Ser Glu Glu Thr Ile 500 505
510 Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser
Ala Gln 515 520 525
Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu 530
535 540 Lys Val Leu Pro Lys
His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr 545 550
555 560 Asn Glu Leu Thr Lys Val Lys Tyr Val Thr
Glu Gly Met Arg Lys Pro 565 570
575 Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
Phe 580 585 590 Lys
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe 595
600 605 Lys Lys Ile Glu Cys Phe
Asp Ser Val Glu Ile Ser Gly Val Glu Asp 610 615
620 Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp
Leu Leu Lys Ile Ile 625 630 635
640 Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu
645 650 655 Asp Ile
Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu 660
665 670 Glu Arg Leu Lys Thr Tyr Ala
His Leu Phe Asp Asp Lys Val Met Lys 675 680
685 Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg
Leu Ser Arg Lys 690 695 700
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp 705
710 715 720 Phe Leu Lys
Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile 725
730 735 His Asp Asp Ser Leu Thr Phe Lys
Glu Asp Ile Gln Lys Ala Gln Val 740 745
750 Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn
Leu Ala Gly 755 760 765
Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp 770
775 780 Glu Leu Val Lys
Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile 785 790
795 800 Glu Met Ala Arg Glu Asn Gln Thr Thr
Gln Lys Gly Gln Lys Asn Ser 805 810
815 Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu
Gly Ser 820 825 830
Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
835 840 845 Lys Leu Tyr Leu
Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp 850
855 860 Gln Glu Leu Asp Ile Asn Arg Leu
Ser Asp Tyr Asp Val Asp His Ile 865 870
875 880 Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp
Asn Lys Val Leu 885 890
895 Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu
900 905 910 Glu Val Val
Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala 915
920 925 Lys Leu Ile Thr Gln Arg Lys Phe
Asp Asn Leu Thr Lys Ala Glu Arg 930 935
940 Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys
Arg Gln Leu 945 950 955
960 Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
965 970 975 Arg Met Asn Thr
Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val 980
985 990 Lys Val Ile Thr Leu Lys Ser Lys
Leu Val Ser Asp Phe Arg Lys Asp 995 1000
1005 Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn
Tyr His His Ala 1010 1015 1020
His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys
1025 1030 1035 Lys Tyr Pro
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys 1040
1045 1050 Val Tyr Asp Val Arg Lys Met Ile
Ala Lys Ser Glu Gln Glu Ile 1055 1060
1065 Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile
Met Asn 1070 1075 1080
Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1085
1090 1095 Arg Pro Leu Ile Glu
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp 1100 1105
1110 Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
Lys Val Leu Ser Met 1115 1120 1125
Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1130 1135 1140 Phe Ser
Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu 1145
1150 1155 Ile Ala Arg Lys Lys Asp Trp
Asp Pro Lys Lys Tyr Gly Gly Phe 1160 1165
1170 Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val
Ala Lys Val 1175 1180 1185
Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu 1190
1195 1200 Gly Ile Thr Ile Met
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile 1205 1210
1215 Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
Val Lys Lys Asp Leu 1220 1225 1230
Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly
1235 1240 1245 Arg Lys
Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn 1250
1255 1260 Glu Leu Ala Leu Pro Ser Lys
Tyr Val Asn Phe Leu Tyr Leu Ala 1265 1270
1275 Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp
Asn Glu Gln 1280 1285 1290
Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile 1295
1300 1305 Ile Glu Gln Ile Ser
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp 1310 1315
1320 Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
Asn Lys His Arg Asp 1325 1330 1335
Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr
1340 1345 1350 Leu Thr
Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr 1355
1360 1365 Thr Ile Asp Arg Lys Arg Tyr
Thr Ser Thr Lys Glu Val Leu Asp 1370 1375
1380 Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr
Glu Thr Arg 1385 1390 1395
Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr 1400
1405 1410 Lys Lys Ala Gly Gln
Ala Lys Lys Lys Lys 1415 1420
502012DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 50gaatgctgcc ctcagacccg
cttcctccct gtccttgtct gtccaaggag aatgaggtct 60cactggtgga tttcggacta
ccctgaggag ctggcacctg agggacaagg ccccccacct 120gcccagctcc agcctctgat
gaggggtggg agagagctac atgaggttgc taagaaagcc 180tcccctgaag gagaccacac
agtgtgtgag gttggagtct ctagcagcgg gttctgtgcc 240cccagggata gtctggctgt
ccaggcactg ctcttgatat aaacaccacc tcctagttat 300gaaaccatgc ccattctgcc
tctctgtatg gaaaagagca tggggctggc ccgtggggtg 360gtgtccactt taggccctgt
gggagatcat gggaacccac gcagtgggtc ataggctctc 420tcatttacta ctcacatcca
ctctgtgaag aagcgattat gatctctcct ctagaaactc 480gtagagtccc atgtctgccg
gcttccagag cctgcactcc tccaccttgg cttggctttg 540ctggggctag aggagctagg
atgcacagca gctctgtgac cctttgtttg agaggaacag 600gaaaaccacc cttctctctg
gcccactgtg tcctcttcct gccctgccat ccccttctgt 660gaatgttaga cccatgggag
cagctggtca gaggggaccc cggcctgggg cccctaaccc 720tatgtagcct cagtcttccc
atcaggctct cagctcagcc tgagtgttga ggccccagtg 780gctgctctgg gggcctcctg
agtttctcat ctgtgcccct ccctccctgg cccaggtgaa 840ggtgtggttc cagaaccgga
ggacaaagta caaacggcag aagctggagg aggaagggcc 900tgagtccgag cagaagaaga
agggctccca tcacatcaac cggtggcgca ttgccacgaa 960gcaggccaat ggggaggaca
tcgatgtcac ctccaatgac aagcttgcta gcggtgggca 1020accacaaacc cacgagggca
gagtgctgct tgctgctggc caggcccctg cgtgggccca 1080agctggactc tggccactcc
ctggccaggc tttggggagg cctggagtca tggccccaca 1140gggcttgaag cccggggccg
ccattgacag agggacaagc aatgggctgg ctgaggcctg 1200ggaccacttg gccttctcct
cggagagcct gcctgcctgg gcgggcccgc ccgccaccgc 1260agcctcccag ctgctctccg
tgtctccaat ctcccttttg ttttgatgca tttctgtttt 1320aatttatttt ccaggcacca
ctgtagttta gtgatcccca gtgtccccct tccctatggg 1380aataataaaa gtctctctct
taatgacacg ggcatccagc tccagcccca gagcctgggg 1440tggtagattc cggctctgag
ggccagtggg ggctggtaga gcaaacgcgt tcagggcctg 1500ggagcctggg gtggggtact
ggtggagggg gtcaagggta attcattaac tcctctcttt 1560tgttggggga ccctggtctc
tacctccagc tccacagcag gagaaacagg ctagacatag 1620ggaagggcca tcctgtatct
tgagggagga caggcccagg tctttcttaa cgtattgaga 1680ggtgggaatc aggcccaggt
agttcaatgg gagagggaga gtgcttccct ctgcctagag 1740actctggtgg cttctccagt
tgaggagaaa ccagaggaaa ggggaggatt ggggtctggg 1800ggagggaaca ccattcacaa
aggctgacgg ttccagtccg aagtcgtggg cccaccagga 1860tgctcacctg tccttggaga
accgctgggc aggttgagac tgcagagaca gggcttaagg 1920ctgagcctgc aaccagtccc
cagtgactca gggcctcctc agcccaagaa agagcaacgt 1980gccagggccc gctgagctct
tgtgttcacc tg 2012511153PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 51Met Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys
Lys Lys 1 5 10 15
Lys Ser Asp Leu Val Leu Gly Leu Asp Ile Gly Ile Gly Ser Val Gly
20 25 30 Val Gly Ile Leu Asn
Lys Val Thr Gly Glu Ile Ile His Lys Asn Ser 35
40 45 Arg Ile Phe Pro Ala Ala Gln Ala Glu
Asn Asn Leu Val Arg Arg Thr 50 55
60 Asn Arg Gln Gly Arg Arg Leu Ala Arg Arg Lys Lys His
Arg Arg Val 65 70 75
80 Arg Leu Asn Arg Leu Phe Glu Glu Ser Gly Leu Ile Thr Asp Phe Thr
85 90 95 Lys Ile Ser Ile
Asn Leu Asn Pro Tyr Gln Leu Arg Val Lys Gly Leu 100
105 110 Thr Asp Glu Leu Ser Asn Glu Glu Leu
Phe Ile Ala Leu Lys Asn Met 115 120
125 Val Lys His Arg Gly Ile Ser Tyr Leu Asp Asp Ala Ser Asp
Asp Gly 130 135 140
Asn Ser Ser Val Gly Asp Tyr Ala Gln Ile Val Lys Glu Asn Ser Lys 145
150 155 160 Gln Leu Glu Thr Lys
Thr Pro Gly Gln Ile Gln Leu Glu Arg Tyr Gln 165
170 175 Thr Tyr Gly Gln Leu Arg Gly Asp Phe Thr
Val Glu Lys Asp Gly Lys 180 185
190 Lys His Arg Leu Ile Asn Val Phe Pro Thr Ser Ala Tyr Arg Ser
Glu 195 200 205 Ala
Leu Arg Ile Leu Gln Thr Gln Gln Glu Phe Asn Pro Gln Ile Thr 210
215 220 Asp Glu Phe Ile Asn Arg
Tyr Leu Glu Ile Leu Thr Gly Lys Arg Lys 225 230
235 240 Tyr Tyr His Gly Pro Gly Asn Glu Lys Ser Arg
Thr Asp Tyr Gly Arg 245 250
255 Tyr Arg Thr Ser Gly Glu Thr Leu Asp Asn Ile Phe Gly Ile Leu Ile
260 265 270 Gly Lys
Cys Thr Phe Tyr Pro Asp Glu Phe Arg Ala Ala Lys Ala Ser 275
280 285 Tyr Thr Ala Gln Glu Phe Asn
Leu Leu Asn Asp Leu Asn Asn Leu Thr 290 295
300 Val Pro Thr Glu Thr Lys Lys Leu Ser Lys Glu Gln
Lys Asn Gln Ile 305 310 315
320 Ile Asn Tyr Val Lys Asn Glu Lys Ala Met Gly Pro Ala Lys Leu Phe
325 330 335 Lys Tyr Ile
Ala Lys Leu Leu Ser Cys Asp Val Ala Asp Ile Lys Gly 340
345 350 Tyr Arg Ile Asp Lys Ser Gly Lys
Ala Glu Ile His Thr Phe Glu Ala 355 360
365 Tyr Arg Lys Met Lys Thr Leu Glu Thr Leu Asp Ile Glu
Gln Met Asp 370 375 380
Arg Glu Thr Leu Asp Lys Leu Ala Tyr Val Leu Thr Leu Asn Thr Glu 385
390 395 400 Arg Glu Gly Ile
Gln Glu Ala Leu Glu His Glu Phe Ala Asp Gly Ser 405
410 415 Phe Ser Gln Lys Gln Val Asp Glu Leu
Val Gln Phe Arg Lys Ala Asn 420 425
430 Ser Ser Ile Phe Gly Lys Gly Trp His Asn Phe Ser Val Lys
Leu Met 435 440 445
Met Glu Leu Ile Pro Glu Leu Tyr Glu Thr Ser Glu Glu Gln Met Thr 450
455 460 Ile Leu Thr Arg Leu
Gly Lys Gln Lys Thr Thr Ser Ser Ser Asn Lys 465 470
475 480 Thr Lys Tyr Ile Asp Glu Lys Leu Leu Thr
Glu Glu Ile Tyr Asn Pro 485 490
495 Val Val Ala Lys Ser Val Arg Gln Ala Ile Lys Ile Val Asn Ala
Ala 500 505 510 Ile
Lys Glu Tyr Gly Asp Phe Asp Asn Ile Val Ile Glu Met Ala Arg 515
520 525 Glu Thr Asn Glu Asp Asp
Glu Lys Lys Ala Ile Gln Lys Ile Gln Lys 530 535
540 Ala Asn Lys Asp Glu Lys Asp Ala Ala Met Leu
Lys Ala Ala Asn Gln 545 550 555
560 Tyr Asn Gly Lys Ala Glu Leu Pro His Ser Val Phe His Gly His Lys
565 570 575 Gln Leu
Ala Thr Lys Ile Arg Leu Trp His Gln Gln Gly Glu Arg Cys 580
585 590 Leu Tyr Thr Gly Lys Thr Ile
Ser Ile His Asp Leu Ile Asn Asn Ser 595 600
605 Asn Gln Phe Glu Val Asp His Ile Leu Pro Leu Ser
Ile Thr Phe Asp 610 615 620
Asp Ser Leu Ala Asn Lys Val Leu Val Tyr Ala Thr Ala Asn Gln Glu 625
630 635 640 Lys Gly Gln
Arg Thr Pro Tyr Gln Ala Leu Asp Ser Met Asp Asp Ala 645
650 655 Trp Ser Phe Arg Glu Leu Lys Ala
Phe Val Arg Glu Ser Lys Thr Leu 660 665
670 Ser Asn Lys Lys Lys Glu Tyr Leu Leu Thr Glu Glu Asp
Ile Ser Lys 675 680 685
Phe Asp Val Arg Lys Lys Phe Ile Glu Arg Asn Leu Val Asp Thr Arg 690
695 700 Tyr Ala Ser Arg
Val Val Leu Asn Ala Leu Gln Glu His Phe Arg Ala 705 710
715 720 His Lys Ile Asp Thr Lys Val Ser Val
Val Arg Gly Gln Phe Thr Ser 725 730
735 Gln Leu Arg Arg His Trp Gly Ile Glu Lys Thr Arg Asp Thr
Tyr His 740 745 750
His His Ala Val Asp Ala Leu Ile Ile Ala Ala Ser Ser Gln Leu Asn
755 760 765 Leu Trp Lys Lys
Gln Lys Asn Thr Leu Val Ser Tyr Ser Glu Asp Gln 770
775 780 Leu Leu Asp Ile Glu Thr Gly Glu
Leu Ile Ser Asp Asp Glu Tyr Lys 785 790
795 800 Glu Ser Val Phe Lys Ala Pro Tyr Gln His Phe Val
Asp Thr Leu Lys 805 810
815 Ser Lys Glu Phe Glu Asp Ser Ile Leu Phe Ser Tyr Gln Val Asp Ser
820 825 830 Lys Phe Asn
Arg Lys Ile Ser Asp Ala Thr Ile Tyr Ala Thr Arg Gln 835
840 845 Ala Lys Val Gly Lys Asp Lys Ala
Asp Glu Thr Tyr Val Leu Gly Lys 850 855
860 Ile Lys Asp Ile Tyr Thr Gln Asp Gly Tyr Asp Ala Phe
Met Lys Ile 865 870 875
880 Tyr Lys Lys Asp Lys Ser Lys Phe Leu Met Tyr Arg His Asp Pro Gln
885 890 895 Thr Phe Glu Lys
Val Ile Glu Pro Ile Leu Glu Asn Tyr Pro Asn Lys 900
905 910 Gln Ile Asn Glu Lys Gly Lys Glu Val
Pro Cys Asn Pro Phe Leu Lys 915 920
925 Tyr Lys Glu Glu His Gly Tyr Ile Arg Lys Tyr Ser Lys Lys
Gly Asn 930 935 940
Gly Pro Glu Ile Lys Ser Leu Lys Tyr Tyr Asp Ser Lys Leu Gly Asn 945
950 955 960 His Ile Asp Ile Thr
Pro Lys Asp Ser Asn Asn Lys Val Val Leu Gln 965
970 975 Ser Val Ser Pro Trp Arg Ala Asp Val Tyr
Phe Asn Lys Thr Thr Gly 980 985
990 Lys Tyr Glu Ile Leu Gly Leu Lys Tyr Ala Asp Leu Gln Phe
Glu Lys 995 1000 1005
Gly Thr Gly Thr Tyr Lys Ile Ser Gln Glu Lys Tyr Asn Asp Ile 1010
1015 1020 Lys Lys Lys Glu Gly
Val Asp Ser Asp Ser Glu Phe Lys Phe Thr 1025 1030
1035 Leu Tyr Lys Asn Asp Leu Leu Leu Val Lys
Asp Thr Glu Thr Lys 1040 1045 1050
Glu Gln Gln Leu Phe Arg Phe Leu Ser Arg Thr Met Pro Lys Gln
1055 1060 1065 Lys His
Tyr Val Glu Leu Lys Pro Tyr Asp Lys Gln Lys Phe Glu 1070
1075 1080 Gly Gly Glu Ala Leu Ile Lys
Val Leu Gly Asn Val Ala Asn Ser 1085 1090
1095 Gly Gln Cys Lys Lys Gly Leu Gly Lys Ser Asn Ile
Ser Ile Tyr 1100 1105 1110
Lys Val Arg Thr Asp Val Leu Gly Asn Gln His Ile Ile Lys Asn 1115
1120 1125 Glu Gly Asp Lys Pro
Lys Leu Asp Phe Lys Arg Pro Ala Ala Thr 1130 1135
1140 Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
1145 1150 52340DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 52gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
tgttagagag 60ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
gtgacgtaga 120aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
ggactatcat 180atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
gtggaaagga 240cgaaacaccg ttacttaaat cttgcagaag ctacaaagat aaggcttcat
gccgaaatca 300acaccctgtc attttatggc agggtgtttt cgttatttaa
34053360DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 53gagggcctat
ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60ataattggaa
ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120aagtaataat
ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180atgcttaccg
taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240cgaaacaccg
ggttttagag ctatgctgtt ttgaatggtc ccaaaacnnn nnnnnnnnnn 300nnnnnnnnnn
nnnnnnngtt ttagagctat gctgttttga atggtcccaa aacttttttt
36054318DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 54gagggcctat ttcccatgat
tccttcatat ttgcatatac gatacaaggc tgttagagag 60ataattggaa ttaatttgac
tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120aagtaataat ttcttgggta
gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180atgcttaccg taacttgaaa
gtatttcgat ttcttggctt tatatatctt gtggaaagga 240cgaaacaccn nnnnnnnnnn
nnnnnnnnng ttttagagct agaaatagca agttaaaata 300aggctagtcc gttttttt
31855325DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 55gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
tgttagagag 60ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
gtgacgtaga 120aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
ggactatcat 180atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
gtggaaagga 240cgaaacaccn nnnnnnnnnn nnnnnnnnng ttttagagct agaaatagca
agttaaaata 300aggctagtcc gttatcattt ttttt
32556337DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 56gagggcctat
ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60ataattggaa
ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120aagtaataat
ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180atgcttaccg
taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240cgaaacaccn
nnnnnnnnnn nnnnnnnnng ttttagagct agaaatagca agttaaaata 300aggctagtcc
gttatcaact tgaaaaagtg ttttttt
33757352DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 57gagggcctat ttcccatgat
tccttcatat ttgcatatac gatacaaggc tgttagagag 60ataattggaa ttaatttgac
tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120aagtaataat ttcttgggta
gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180atgcttaccg taacttgaaa
gtatttcgat ttcttggctt tatatatctt gtggaaagga 240cgaaacaccn nnnnnnnnnn
nnnnnnnnng ttttagagct agaaatagca agttaaaata 300aggctagtcc gttatcaact
tgaaaaagtg gcaccgagtc ggtgcttttt tt 352585101DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 58cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
cccgcccatt 60gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
attgacgtca 120atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
atcatatgcc 180aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
atgcccagta 240catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
tcgctattac 300catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
cctccccacc 360cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
cggggggggg 420gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
cgaggcggag 480aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
tggcgaggcg 540gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
ctgcgacgct 600gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
ctctgactga 660ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
tgtaattagc 720tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg
tttaattacc 780tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca
tggactataa 840ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg
acgataagat 900ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg
acaagaagta 960cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca
ccgacgagta 1020caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
gcatcaagaa 1080gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca
cccggctgaa 1140gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
tgcaagagat 1200cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
aagagtcctt 1260cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
tcgtggacga 1320ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
tggtggacag 1380caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
tcaagttccg 1440gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
acaagctgtt 1500catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
acgccagcgg 1560cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
tggaaaatct 1620gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga
ttgccctgag 1680cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
ccaaactgca 1740gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
tcggcgacca 1800gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
tgagcgacat 1860cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
tcaagagata 1920cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
agctgcctga 1980gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
acattgacgg 2040cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
agatggacgg 2100caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
agcggacctt 2160cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
ttctgcggcg 2220gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
agatcctgac 2280cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
tcgcctggat 2340gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
tggacaaggg 2400cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
tgcccaacga 2460gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata
acgagctgac 2520caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
gcgagcagaa 2580aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
agcagctgaa 2640agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
gcgtggaaga 2700tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
aggacaagga 2760cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
ccctgacact 2820gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
tgttcgacga 2880caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
tgagccggaa 2940gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
tcctgaagtc 3000cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
tgacctttaa 3060agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
agcacattgc 3120caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
aggtggtgga 3180cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
aaatggccag 3240agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
agcggatcga 3300agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
aaaacaccca 3360gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
tgtacgtgga 3420ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg
tgcctcagag 3480ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca
agaaccgggg 3540caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
actggcggca 3600gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca
aggccgagag 3660aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
tggaaacccg 3720gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
agtacgacga 3780gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
tggtgtccga 3840tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
accacgccca 3900cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
ctaagctgga 3960aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
tcgccaagag 4020cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
tcatgaactt 4080tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
tgatcgagac 4140aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
ccgtgcggaa 4200agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
caggcggctt 4260cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca
gaaagaagga 4320ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
ctgtgctggt 4380ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
agctgctggg 4440gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
tggaagccaa 4500gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
ccctgttcga 4560gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
agggaaacga 4620actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
atgagaagct 4680gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
acaagcacta 4740cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
tggccgacgc 4800taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca
tcagagagca 4860ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
ccgccttcaa 4920gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
tgctggacgc 4980caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
tgtctcagct 5040gggaggcgac tttctttttc ttagcttgac cagctttctt agtagcagca
ggacgcttta 5100a
510159137DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 59nnnnnnnnnn
nnnnnnnnnn gtttttgtac tctcaagatt tagaaataaa tcttgcagaa 60gctacaaaga
taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt 120tcgttattta
atttttt
13760123DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 60nnnnnnnnnn nnnnnnnnnn
gtttttgtac tctcagaaat gcagaagcta caaagataag 60gcttcatgcc gaaatcaaca
ccctgtcatt ttatggcagg gtgttttcgt tatttaattt 120ttt
12361110DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 61nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcagaaat gcagaagcta
caaagataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttttt
11062137DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 62nnnnnnnnnn
nnnnnnnnnn gttattgtac tctcaagatt tagaaataaa tcttgcagaa 60gctacaaaga
taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt 120tcgttattta
atttttt
13763123DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 63nnnnnnnnnn nnnnnnnnnn
gttattgtac tctcagaaat gcagaagcta caaagataag 60gcttcatgcc gaaatcaaca
ccctgtcatt ttatggcagg gtgttttcgt tatttaattt 120ttt
12364110DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 64nnnnnnnnnn nnnnnnnnnn gttattgtac tctcagaaat gcagaagcta
caaagataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttttt
11065137DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 65nnnnnnnnnn
nnnnnnnnnn gttattgtac tctcaagatt tagaaataaa tcttgcagaa 60gctacaatga
taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt 120tcgttattta
atttttt
13766123DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 66nnnnnnnnnn nnnnnnnnnn
gttattgtac tctcagaaat gcagaagcta caatgataag 60gcttcatgcc gaaatcaaca
ccctgtcatt ttatggcagg gtgttttcgt tatttaattt 120ttt
12367110DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 67nnnnnnnnnn nnnnnnnnnn gttattgtac tctcagaaat gcagaagcta
caatgataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttttt
11068107DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 68nnnnnnnnnn
nnnnnnnnnn gttttagagc tgtggaaaca cagcgagtta aaataaggct 60tagtccgtac
tcaacttgaa aaggtggcac cgattcggtg ttttttt
107694263DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 69atgaaaaggc cggcggccac
gaaaaaggcc ggccaggcaa aaaagaaaaa gaccaagccc 60tacagcatcg gcctggacat
cggcaccaat agcgtgggct gggccgtgac caccgacaac 120tacaaggtgc ccagcaagaa
aatgaaggtg ctgggcaaca cctccaagaa gtacatcaag 180aaaaacctgc tgggcgtgct
gctgttcgac agcggcatta cagccgaggg cagacggctg 240aagagaaccg ccagacggcg
gtacacccgg cggagaaaca gaatcctgta tctgcaagag 300atcttcagca ccgagatggc
taccctggac gacgccttct tccagcggct ggacgacagc 360ttcctggtgc ccgacgacaa
gcgggacagc aagtacccca tcttcggcaa cctggtggaa 420gagaaggcct accacgacga
gttccccacc atctaccacc tgagaaagta cctggccgac 480agcaccaaga aggccgacct
gagactggtg tatctggccc tggcccacat gatcaagtac 540cggggccact tcctgatcga
gggcgagttc aacagcaaga acaacgacat ccagaagaac 600ttccaggact tcctggacac
ctacaacgcc atcttcgaga gcgacctgtc cctggaaaac 660agcaagcagc tggaagagat
cgtgaaggac aagatcagca agctggaaaa gaaggaccgc 720atcctgaagc tgttccccgg
cgagaagaac agcggaatct tcagcgagtt tctgaagctg 780atcgtgggca accaggccga
cttcagaaag tgcttcaacc tggacgagaa agccagcctg 840cacttcagca aagagagcta
cgacgaggac ctggaaaccc tgctgggata tatcggcgac 900gactacagcg acgtgttcct
gaaggccaag aagctgtacg acgctatcct gctgagcggc 960ttcctgaccg tgaccgacaa
cgagacagag gccccactga gcagcgccat gattaagcgg 1020tacaacgagc acaaagagga
tctggctctg ctgaaagagt acatccggaa catcagcctg 1080aaaacctaca atgaggtgtt
caaggacgac accaagaacg gctacgccgg ctacatcgac 1140ggcaagacca accaggaaga
tttctatgtg tacctgaaga agctgctggc cgagttcgag 1200ggggccgact actttctgga
aaaaatcgac cgcgaggatt tcctgcggaa gcagcggacc 1260ttcgacaacg gcagcatccc
ctaccagatc catctgcagg aaatgcgggc catcctggac 1320aagcaggcca agttctaccc
attcctggcc aagaacaaag agcggatcga gaagatcctg 1380accttccgca tcccttacta
cgtgggcccc ctggccagag gcaacagcga ttttgcctgg 1440tccatccgga agcgcaatga
gaagatcacc ccctggaact tcgaggacgt gatcgacaaa 1500gagtccagcg ccgaggcctt
catcaaccgg atgaccagct tcgacctgta cctgcccgag 1560gaaaaggtgc tgcccaagca
cagcctgctg tacgagacat tcaatgtgta taacgagctg 1620accaaagtgc ggtttatcgc
cgagtctatg cgggactacc agttcctgga ctccaagcag 1680aaaaaggaca tcgtgcggct
gtacttcaag gacaagcgga aagtgaccga taaggacatc 1740atcgagtacc tgcacgccat
ctacggctac gatggcatcg agctgaaggg catcgagaag 1800cagttcaact ccagcctgag
cacataccac gacctgctga acattatcaa cgacaaagaa 1860tttctggacg actccagcaa
cgaggccatc atcgaagaga tcatccacac cctgaccatc 1920tttgaggacc gcgagatgat
caagcagcgg ctgagcaagt tcgagaacat cttcgacaag 1980agcgtgctga aaaagctgag
cagacggcac tacaccggct ggggcaagct gagcgccaag 2040ctgatcaacg gcatccggga
cgagaagtcc ggcaacacaa tcctggacta cctgatcgac 2100gacggcatca gcaaccggaa
cttcatgcag ctgatccacg acgacgccct gagcttcaag 2160aagaagatcc agaaggccca
gatcatcggg gacgaggaca agggcaacat caaagaagtc 2220gtgaagtccc tgcccggcag
ccccgccatc aagaagggaa tcctgcagag catcaagatc 2280gtggacgagc tcgtgaaagt
gatgggcggc agaaagcccg agagcatcgt ggtggaaatg 2340gctagagaga accagtacac
caatcagggc aagagcaaca gccagcagag actgaagaga 2400ctggaaaagt ccctgaaaga
gctgggcagc aagattctga aagagaatat ccctgccaag 2460ctgtccaaga tcgacaacaa
cgccctgcag aacgaccggc tgtacctgta ctacctgcag 2520aatggcaagg acatgtatac
aggcgacgac ctggatatcg accgcctgag caactacgac 2580atcgaccata ttatccccca
ggccttcctg aaagacaaca gcattgacaa caaagtgctg 2640gtgtcctccg ccagcaaccg
cggcaagtcc gatgatgtgc ccagcctgga agtcgtgaaa 2700aagagaaaga ccttctggta
tcagctgctg aaaagcaagc tgattagcca gaggaagttc 2760gacaacctga ccaaggccga
gagaggcggc ctgagccctg aagataaggc cggcttcatc 2820cagagacagc tggtggaaac
ccggcagatc accaagcacg tggccagact gctggatgag 2880aagtttaaca acaagaagga
cgagaacaac cgggccgtgc ggaccgtgaa gatcatcacc 2940ctgaagtcca ccctggtgtc
ccagttccgg aaggacttcg agctgtataa agtgcgcgag 3000atcaatgact ttcaccacgc
ccacgacgcc tacctgaatg ccgtggtggc ttccgccctg 3060ctgaagaagt accctaagct
ggaacccgag ttcgtgtacg gcgactaccc caagtacaac 3120tccttcagag agcggaagtc
cgccaccgag aaggtgtact tctactccaa catcatgaat 3180atctttaaga agtccatctc
cctggccgat ggcagagtga tcgagcggcc cctgatcgaa 3240gtgaacgaag agacaggcga
gagcgtgtgg aacaaagaaa gcgacctggc caccgtgcgg 3300cgggtgctga gttatcctca
agtgaatgtc gtgaagaagg tggaagaaca gaaccacggc 3360ctggatcggg gcaagcccaa
gggcctgttc aacgccaacc tgtccagcaa gcctaagccc 3420aactccaacg agaatctcgt
gggggccaaa gagtacctgg accctaagaa gtacggcgga 3480tacgccggca tctccaatag
cttcaccgtg ctcgtgaagg gcacaatcga gaagggcgct 3540aagaaaaaga tcacaaacgt
gctggaattt caggggatct ctatcctgga ccggatcaac 3600taccggaagg ataagctgaa
ctttctgctg gaaaaaggct acaaggacat tgagctgatt 3660atcgagctgc ctaagtactc
cctgttcgaa ctgagcgacg gctccagacg gatgctggcc 3720tccatcctgt ccaccaacaa
caagcggggc gagatccaca agggaaacca gatcttcctg 3780agccagaaat ttgtgaaact
gctgtaccac gccaagcgga tctccaacac catcaatgag 3840aaccaccgga aatacgtgga
aaaccacaag aaagagtttg aggaactgtt ctactacatc 3900ctggagttca acgagaacta
tgtgggagcc aagaagaacg gcaaactgct gaactccgcc 3960ttccagagct ggcagaacca
cagcatcgac gagctgtgca gctccttcat cggccctacc 4020ggcagcgagc ggaagggact
gtttgagctg acctccagag gctctgccgc cgactttgag 4080ttcctgggag tgaagatccc
ccggtacaga gactacaccc cctctagtct gctgaaggac 4140gccaccctga tccaccagag
cgtgaccggc ctgtacgaaa cccggatcga cctggctaag 4200ctgggcgagg gaaagcgtcc
tgctgctact aagaaagctg gtcaagctaa gaaaaagaaa 4260taa
42637084DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 70ggaaccattc ataacagcat agcaagttat aataaggcta gtccgttatc
aacttgaaaa 60agtggcaccg agtcggtgct tttt
847136DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 71gttatagagc
tatgctgtta tgaatggtcc caaaac
367284DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 72ggaaccattc aatacagcat
agcaagttaa tataaggcta gtccgttatc aacttgaaaa 60agtggcaccg agtcggtgct
tttt 847336DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 73gtattagagc tatgctgtat tgaatggtcc caaaac
3674103DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 74nnnnnnnnnn
nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc 60cgttatcaac
ttgaaaaagt ggcaccgagt cggtgctttt ttt
10375103DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 75nnnnnnnnnn nnnnnnnnnn
gtattagagc tagaaatagc aagttaatat aaggctagtc 60cgttatcaac ttgaaaaagt
ggcaccgagt cggtgctttt ttt 10376123DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 76nnnnnnnnnn nnnnnnnnnn gttttagagc tatgctgttt tggaaacaaa
acagcatagc 60aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
cggtgctttt 120ttt
12377123DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 77nnnnnnnnnn
nnnnnnnnnn gtattagagc tatgctgtat tggaaacaat acagcatagc 60aagttaatat
aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 120ttt
1237820DNAHomo
sapiens 78gtcacctcca atgactaggg
2079984PRTCampylobacter jejuni 79Met Ala Arg Ile Leu Ala Phe Asp
Ile Gly Ile Ser Ser Ile Gly Trp 1 5 10
15 Ala Phe Ser Glu Asn Asp Glu Leu Lys Asp Cys Gly Val
Arg Ile Phe 20 25 30
Thr Lys Val Glu Asn Pro Lys Thr Gly Glu Ser Leu Ala Leu Pro Arg
35 40 45 Arg Leu Ala Arg
Ser Ala Arg Lys Arg Leu Ala Arg Arg Lys Ala Arg 50
55 60 Leu Asn His Leu Lys His Leu Ile
Ala Asn Glu Phe Lys Leu Asn Tyr 65 70
75 80 Glu Asp Tyr Gln Ser Phe Asp Glu Ser Leu Ala Lys
Ala Tyr Lys Gly 85 90
95 Ser Leu Ile Ser Pro Tyr Glu Leu Arg Phe Arg Ala Leu Asn Glu Leu
100 105 110 Leu Ser Lys
Gln Asp Phe Ala Arg Val Ile Leu His Ile Ala Lys Arg 115
120 125 Arg Gly Tyr Asp Asp Ile Lys Asn
Ser Asp Asp Lys Glu Lys Gly Ala 130 135
140 Ile Leu Lys Ala Ile Lys Gln Asn Glu Glu Lys Leu Ala
Asn Tyr Gln 145 150 155
160 Ser Val Gly Glu Tyr Leu Tyr Lys Glu Tyr Phe Gln Lys Phe Lys Glu
165 170 175 Asn Ser Lys Glu
Phe Thr Asn Val Arg Asn Lys Lys Glu Ser Tyr Glu 180
185 190 Arg Cys Ile Ala Gln Ser Phe Leu Lys
Asp Glu Leu Lys Leu Ile Phe 195 200
205 Lys Lys Gln Arg Glu Phe Gly Phe Ser Phe Ser Lys Lys Phe
Glu Glu 210 215 220
Glu Val Leu Ser Val Ala Phe Tyr Lys Arg Ala Leu Lys Asp Phe Ser 225
230 235 240 His Leu Val Gly Asn
Cys Ser Phe Phe Thr Asp Glu Lys Arg Ala Pro 245
250 255 Lys Asn Ser Pro Leu Ala Phe Met Phe Val
Ala Leu Thr Arg Ile Ile 260 265
270 Asn Leu Leu Asn Asn Leu Lys Asn Thr Glu Gly Ile Leu Tyr Thr
Lys 275 280 285 Asp
Asp Leu Asn Ala Leu Leu Asn Glu Val Leu Lys Asn Gly Thr Leu 290
295 300 Thr Tyr Lys Gln Thr Lys
Lys Leu Leu Gly Leu Ser Asp Asp Tyr Glu 305 310
315 320 Phe Lys Gly Glu Lys Gly Thr Tyr Phe Ile Glu
Phe Lys Lys Tyr Lys 325 330
335 Glu Phe Ile Lys Ala Leu Gly Glu His Asn Leu Ser Gln Asp Asp Leu
340 345 350 Asn Glu
Ile Ala Lys Asp Ile Thr Leu Ile Lys Asp Glu Ile Lys Leu 355
360 365 Lys Lys Ala Leu Ala Lys Tyr
Asp Leu Asn Gln Asn Gln Ile Asp Ser 370 375
380 Leu Ser Lys Leu Glu Phe Lys Asp His Leu Asn Ile
Ser Phe Lys Ala 385 390 395
400 Leu Lys Leu Val Thr Pro Leu Met Leu Glu Gly Lys Lys Tyr Asp Glu
405 410 415 Ala Cys Asn
Glu Leu Asn Leu Lys Val Ala Ile Asn Glu Asp Lys Lys 420
425 430 Asp Phe Leu Pro Ala Phe Asn Glu
Thr Tyr Tyr Lys Asp Glu Val Thr 435 440
445 Asn Pro Val Val Leu Arg Ala Ile Lys Glu Tyr Arg Lys
Val Leu Asn 450 455 460
Ala Leu Leu Lys Lys Tyr Gly Lys Val His Lys Ile Asn Ile Glu Leu 465
470 475 480 Ala Arg Glu Val
Gly Lys Asn His Ser Gln Arg Ala Lys Ile Glu Lys 485
490 495 Glu Gln Asn Glu Asn Tyr Lys Ala Lys
Lys Asp Ala Glu Leu Glu Cys 500 505
510 Glu Lys Leu Gly Leu Lys Ile Asn Ser Lys Asn Ile Leu Lys
Leu Arg 515 520 525
Leu Phe Lys Glu Gln Lys Glu Phe Cys Ala Tyr Ser Gly Glu Lys Ile 530
535 540 Lys Ile Ser Asp Leu
Gln Asp Glu Lys Met Leu Glu Ile Asp His Ile 545 550
555 560 Tyr Pro Tyr Ser Arg Ser Phe Asp Asp Ser
Tyr Met Asn Lys Val Leu 565 570
575 Val Phe Thr Lys Gln Asn Gln Glu Lys Leu Asn Gln Thr Pro Phe
Glu 580 585 590 Ala
Phe Gly Asn Asp Ser Ala Lys Trp Gln Lys Ile Glu Val Leu Ala 595
600 605 Lys Asn Leu Pro Thr Lys
Lys Gln Lys Arg Ile Leu Asp Lys Asn Tyr 610 615
620 Lys Asp Lys Glu Gln Lys Asn Phe Lys Asp Arg
Asn Leu Asn Asp Thr 625 630 635
640 Arg Tyr Ile Ala Arg Leu Val Leu Asn Tyr Thr Lys Asp Tyr Leu Asp
645 650 655 Phe Leu
Pro Leu Ser Asp Asp Glu Asn Thr Lys Leu Asn Asp Thr Gln 660
665 670 Lys Gly Ser Lys Val His Val
Glu Ala Lys Ser Gly Met Leu Thr Ser 675 680
685 Ala Leu Arg His Thr Trp Gly Phe Ser Ala Lys Asp
Arg Asn Asn His 690 695 700
Leu His His Ala Ile Asp Ala Val Ile Ile Ala Tyr Ala Asn Asn Ser 705
710 715 720 Ile Val Lys
Ala Phe Ser Asp Phe Lys Lys Glu Gln Glu Ser Asn Ser 725
730 735 Ala Glu Leu Tyr Ala Lys Lys Ile
Ser Glu Leu Asp Tyr Lys Asn Lys 740 745
750 Arg Lys Phe Phe Glu Pro Phe Ser Gly Phe Arg Gln Lys
Val Leu Asp 755 760 765
Lys Ile Asp Glu Ile Phe Val Ser Lys Pro Glu Arg Lys Lys Pro Ser 770
775 780 Gly Ala Leu His
Glu Glu Thr Phe Arg Lys Glu Glu Glu Phe Tyr Gln 785 790
795 800 Ser Tyr Gly Gly Lys Glu Gly Val Leu
Lys Ala Leu Glu Leu Gly Lys 805 810
815 Ile Arg Lys Val Asn Gly Lys Ile Val Lys Asn Gly Asp Met
Phe Arg 820 825 830
Val Asp Ile Phe Lys His Lys Lys Thr Asn Lys Phe Tyr Ala Val Pro
835 840 845 Ile Tyr Thr Met
Asp Phe Ala Leu Lys Val Leu Pro Asn Lys Ala Val 850
855 860 Ala Arg Ser Lys Lys Gly Glu Ile
Lys Asp Trp Ile Leu Met Asp Glu 865 870
875 880 Asn Tyr Glu Phe Cys Phe Ser Leu Tyr Lys Asp Ser
Leu Ile Leu Ile 885 890
895 Gln Thr Lys Asp Met Gln Glu Pro Glu Phe Val Tyr Tyr Asn Ala Phe
900 905 910 Thr Ser Ser
Thr Val Ser Leu Ile Val Ser Lys His Asp Asn Lys Phe 915
920 925 Glu Thr Leu Ser Lys Asn Gln Lys
Ile Leu Phe Lys Asn Ala Asn Glu 930 935
940 Lys Glu Val Ile Ala Lys Ser Ile Gly Ile Gln Asn Leu
Lys Val Phe 945 950 955
960 Glu Lys Tyr Ile Val Ser Ala Leu Gly Glu Val Thr Lys Ala Glu Phe
965 970 975 Arg Gln Arg Glu
Asp Phe Lys Lys 980 8091DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 80tataatctca taagaaattt aaaaagggac taaaataaag agtttgcggg
actctgcggg 60gttacaatcc cctaaaaccg cttttaaaat t
918136DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 81attttaccat
aaagaaattt aaaaagggac taaaac
368295RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 82nnnnnnnnnn nnnnnnnnnn
guuuuagucc cgaaagggac uaaaauaaag aguuugcggg 60acucugcggg guuacaaucc
ccuaaaaccg cuuuu 958369RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 83gucaccucca augacuaggg guuuuagagc uagaaauagc aaguuaaaau
aaggcuaguc 60cguuuuuuu
698469RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 84gacaucgaug
uccuccccau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuuuuuu
698569RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 85gaguccgagc agaagaagaa
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuuuuuu
698669RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 86ggggccgaga uuggguguuc guuuuagagc uagaaauagc aaguuaaaau
aaggcuaguc 60cguuuuuuu
698769RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 87guggcgagag
gggccgagau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuuuuuu
698876RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 88gucaccucca augacuaggg
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu
768976RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 89gacaucgaug uccuccccau guuuuagagc uagaaauagc aaguuaaaau
aaggcuaguc 60cguuaucauu uuuuuu
769076RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 90gaguccgagc
agaagaagaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucauu
uuuuuu
769176RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 91ggggccgaga uuggguguuc
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu
769276RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 92guggcgagag gggccgagau guuuuagagc uagaaauagc aaguuaaaau
aaggcuaguc 60cguuaucauu uuuuuu
769388RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 93gucaccucca
augacuaggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac
uugaaaaagu guuuuuuu
889488RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 94gacaucgaug uccuccccau
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu
guuuuuuu 889588RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 95gaguccgagc agaagaagaa guuuuagagc uagaaauagc aaguuaaaau
aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu
889688RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 96ggggccgaga
uuggguguuc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac
uugaaaaagu guuuuuuu
889788RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 97guggcgagag gggccgagau
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu
guuuuuuu 8898103RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 98gucaccucca augacuaggg guuuuagagc uagaaauagc aaguuaaaau
aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu
10399103RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 99gacaucgaug
uccuccccau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu uuu
103100103RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 100gaguccgagc agaagaagaa
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu
ggcaccgagu cggugcuuuu uuu 103101103RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 101ggggccgaga uuggguguuc guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu
uuu 103102103RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 102guggcgagag gggccgagau guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu
uuu 103103102DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 103gttttagagc tatgctgttt tgaatggtcc caaaacggaa gggcctgagt
ccgagcagaa 60gaagaagttt tagagctatg ctgttttgaa tggtcccaaa ac
102104100DNAHomo sapiens 104cggaggacaa agtacaaacg gcagaagctg
gaggaggaag ggcctgagtc cgagcagaag 60aagaagggct cccatcacat caaccggtgg
cgcattgcca 10010550DNAHomo sapiens 105agctggagga
ggaagggcct gagtccgagc agaagaagaa gggctcccac
5010630RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 106gaguccgagc agaagaagaa
guuuuagagc 3010749DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 107agctggagga ggaagggcct gagtccgagc agaagagaag ggctcccat
4910853DNAHomo sapiens 108ctggaggagg aagggcctga
gtccgagcag aagaagaagg gctcccatca cat 5310952DNAHomo sapiens
109ctggaggagg aagggcctga gtccgagcag aagagaaggg ctcccatcac at
5211054DNAHomo sapiens 110ctggaggagg aagggcctga gtccgagcag aagaaagaag
ggctcccatc acat 5411150DNAHomo sapiens 111ctggaggagg aagggcctga
gtccgagcag aagaagggct cccatcacat 5011247DNAHomo sapiens
112ctggaggagg aagggcctga gcccgagcag aagggctccc atcacat
4711366DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 113nnnnnnnnnn nnnnnnnnnn
guuuuagagc uagaaauagc aaguuaaaau aaggctagtc 60cguuuu
6611420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 114gaguccgagc agaagaagaa
2011520RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 115gacaucgaug
uccuccccau
2011620RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 116gucaccucca augacuaggg
2011720RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 117auuggguguu cagggcagag
2011820RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 118guggcgagag
gggccgagau
2011920RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 119ggggccgaga uuggguguuc
2012020RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 120gugccauuag cuaaaugcau
2012120RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 121guaccaccca
caggugccag
2012220RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 122gaaagccucu gggccaggaa
2012348DNAHomo sapiens
123ctggaggagg aagggcctga gtccgagcag aagaagaagg gctcccat
4812420RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 124gaguccgagc agaagaagau
2012520RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 125gaguccgagc agaagaagua
2012620RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 126gaguccgagc
agaagaacaa
2012720RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 127gaguccgagc agaagaugaa
2012820RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 128gaguccgagc agaaguagaa
2012920RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 129gaguccgagc
agaugaagaa
2013020RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 130gaguccgagc acaagaagaa
2013120RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 131gaguccgagg agaagaagaa
2013220RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 132gaguccgugc
agaagaagaa
2013320RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 133gagucggagc agaagaagaa
2013420RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 134gagaccgagc agaagaagaa
2013524DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 135aatgacaagc
ttgctagcgg tggg
2413639DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 136aaaacggaag ggcctgagtc
cgagcagaag aagaagttt 3913739DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 137aaacaggggc cgagattggg tgttcagggc agaggtttt
3913838DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 138aaaacggaag
ggcctgagtc cgagcagaag aagaagtt
3813940DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 139aacggaggga ggggcacaga
tgagaaactc agggttttag 4014038DNAHomo sapiens
140agcccttctt cttctgctcg gactcaggcc cttcctcc
3814140DNAHomo sapiens 141cagggaggga ggggcacaga tgagaaactc aggaggcccc
4014280DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 142ggcaatgcgc caccggttga tgtgatggga gcccttctag
gaggccccca gagcagccac 60tggggcctca acactcaggc
8014398DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 143ggacgaaaca ccggaaccat tcaaaacagc atagcaagtt
aaaataaggc tagtccgtta 60tcaacttgaa aaagtggcac cgagtcggtg cttttttt
98144186DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 144ggacgaaaca ccggtagtat taagtattgt tttatggctg ataaatttct
ttgaatttct 60ccttgattat ttgttataaa agttataaaa taatcttgtt ggaaccattc
aaaacagcat 120agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
agtcggtgct 180tttttt
18614546RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 145nnnnnnnnnn
nnnnnnnnng uuauuguacu cucaagauuu auuuuu
4614691RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 146guuacuuaaa ucuugcagaa
gcuacaaaga uaaggcuuca ugccgaaauc aacacccugu 60cauuuuaugg caggguguuu
ucguuauuua a 9114770DNAHomo sapiens
147ttttctagtg ctgagtttct gtgactcctc tacattctac ttctctgtgt ttctgtatac
60tacctcctcc
70148122DNAHomo sapiens 148ggaggaaggg cctgagtccg agcagaagaa gaagggctcc
catcacatca accggtggcg 60cattgccacg aagcaggcca atggggagga catcgatgtc
acctccaatg actagggtgg 120gc
12214948RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 149acnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnguuuuaga gcuaugcu
4815067DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 150agcauagcaa
guuaaaauaa ggctaguccg uuaucaacuu gaaaaagugg caccgagucg 60gugcuuu
6715162RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 151nnnnnnnnnn nnnnnnnnnn
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cg
6215273DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 152tgaatggtcc caaaacggaa gggcctgagt ccgagcagaa
gaagaagttt tagagctatg 60ctgttttgaa tgg
7315399RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 153nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuu
99154127RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 154guuuuuguac ucucaagauu uaaguaacug uacaacguua cuuaaaucuu
gcagaagcua 60caaagauaag gcuucaugcc gaaaucaaca cccugucauu uuauggcagg
guguuuucgu 120uauuuaa
12715556DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 155nnnnnnnnnn
nnnnnnnnnn gtttttgtac tctcaagatt taagtaactg tacaac
5615691DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 156gttacttaaa tcttgcagaa
gctacaaaga taaggcttca tgccgaaatc aacaccctgt 60cattttatgg cagggtgttt
tcgttattta a 91157134DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 157nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcaagatt taaggaaact
aaatcttgca 60gaagctacaa agataaggct tcatgccgaa atcaacaccc tgtcatttta
tggcagggtg 120ttttcgttat ttaa
134158131DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 158nnnnnnnnnn
nnnnnnnnnn gtttttgtac tctcaagatt tagaaataaa tcttgcagaa 60gctacaaaga
taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt 120tcgttattta a
131159125DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 159nnnnnnnnnn nnnnnnnnnn
gtttttgtac tctcaagatg aaaatcttgc agaagctaca 60aagataaggc ttcatgccga
aatcaacacc ctgtcatttt atggcagggt gttttcgtta 120tttaa
125160112DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 160nnnnnnnnnn nnnnnnnnnn gtttttgtac tctgaaaaga agctacaaag
ataaggcttc 60atgccgaaat caacaccctg tcattttatg gcagggtgtt ttcgttattt
aa 112161107DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 161nnnnnnnnnn
nnnnnnnnnn gtttttgtac tgaaaagcta caaagataag gcttcatgcc 60gaaatcaaca
ccctgtcatt ttatggcagg gtgttttcgt tatttaa
107162108DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 162nnnnnnnnnn nnnnnnnnnn
gtttttgtac tctcaagatt tagaaataaa tcttgcagaa 60gctacaaaga taaggcttca
tgccgaaatc aacaccctgt cattttat 10816386DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 163nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcaagatt
tagaaataaa tcttgcagaa 60gctacaaaga taaggcttca tgccga
8616479DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 164nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcaagatt
tagaaataaa tcttgcagaa 60gctacaaaga taaggcttc
7916573DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 165nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcaagatt
tagaaataaa tcttgcagaa 60gctacaaaga taa
73166125RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 166guuuuagucc cuuuuuaaau uucuuuaugg uaaaauuaua aucucauaag
aaauuuaaaa 60agggacuaaa auaaagaguu ugcgggacuc ugcgggguua caauccccua
aaaccgcuuu 120uaaaa
12516791RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 167guuacuuaaa
ucuugcagaa gcuacaaaga uaaggcuuca ugccgaaauc aacacccugu 60cauuuuaugg
caggguguuu ucguuauuua a
9116856RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 168gggacucaac caagucauuc
guuuuuguac ucucaagauu uaaguaacug uacaac 56169147RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 169gggacucaac caagucauuc guuuuuguac ucucaagauu uaaguaacug
uacaacguua 60cuuaaaucuu gcagaagcua caaagauaag gcuucaugcc gaaaucaaca
cccugucauu 120uuauggcagg guguuuucgu uauuuaa
14717070RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 170cuugcagaag
cuacaaagau aaggcuucau gccgaaauca acacccuguc auuuuauggc 60aggguguuuu
7017142RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 171gggacucaac caagucauuc
guuuuuguac ucucaagauu ua 42172112RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 172gggacucaac caagucauuc guuuuuguac ucucaagauu uacuugcaga
agcuacaaag 60auaaggcuuc augccgaaau caacacccug ucauuuuaug gcaggguguu
uu 112173116RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 173gggacucaac
caagucauuc guuuuuguac ucucaagauu uagaaacuug cagaagcuac 60aaagauaagg
cuucaugccg aaaucaacac ccugucauuu uauggcaggg uguuuu
116174116RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 174gggacucaac caagucauuc
guuuuuguac ucucaagauu uagaaacuug cagaagcuac 60aaagauaagg cuucaugccg
aaaucaacac ccugucauuu uauggcaggg uguuuu 116175102RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 175gggacucaac caagucauuc guuuuuguag aaauacaaag auaaggcuuc
augccgaaau 60caacacccug ucauuuuaug gcaggguguu uucguuauuu aa
102176102RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 176gggacucaac
caagucauuc guuuuuguag aaauacaaag auaaggcuuc augccgaaau 60caacacccug
ucauuuuaug gcaggguguu uucguuauuu aa
10217757RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 177gggacucaac caagucauuc
guuuuuguag aaauacaaag auaaggcuuc augccga 5717857RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 178gggacucaac caagucauuc guuuuuguag aaauacaaag
auaaggcuuc augccga 5717923DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 179gtggtgtcac gctcgtcgtt tgg
2318023DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 180tccagtctat
taattgttgc cgg 2318164DNAHomo
sapiens 181caagaggctt gagtaggaga ggagtgccgc cgaggcgggg cggggcgggg
cgtggagctg 60ggct
6418299RNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 182nnnnnnnnnn
nnnnnnnnnn guauuagagc uagaaauagc aaguuaauau aaggcuaguc 60cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuu
99183119RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 183nnnnnnnnnn nnnnnnnnnn
guuuuagagc uaugcuguuu uggaaacaaa acagcauagc 60aaguuaaaau aaggcuaguc
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuu 119184119RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 184nnnnnnnnnn nnnnnnnnnn guauuagagc uaugcuguau uggaaacaau
acagcauagc 60aaguuaauau aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu
cggugcuuu 11918512DNAHomo sapiens 185tagcgggtaa gc
1218612DNAHomo sapiens 186tcggtgacat
gt 1218712DNAHomo
sapiens 187actccccgta gg
1218812DNAHomo sapiens 188actgcgtgtt aa
1218912DNAHomo sapiens 189acgtcgcctg at
1219012DNAHomo sapiens
190taggtcgacc ag
1219112DNAHomo sapiens 191ggcgttaatg at
1219212DNAHomo sapiens 192tgtcgcatgt ta
1219312DNAHomo sapiens
193atggaaacgc at
1219412DNAHomo sapiens 194gccgaattcc tc
1219512DNAHomo sapiens 195gcatggtacg ga
1219612DNAHomo sapiens
196cggtactctt ac
1219712DNAHomo sapiens 197gcctgtgccg ta
1219812DNAHomo sapiens 198tacggtaagt cg
1219912DNAHomo sapiens
199cacgaaatta cc
1220012DNAHomo sapiens 200aaccaagata cg
1220112DNAHomo sapiens 201gagtcgatac gc
1220212DNAHomo sapiens
202gtctcacgat cg
1220312DNAHomo sapiens 203tcgtcgggtg ca
1220412DNAHomo sapiens 204actccgtagt ga
1220512DNAHomo sapiens
205caggacgtcc gt
1220612DNAHomo sapiens 206tcgtatccct ac
1220712DNAHomo sapiens 207tttcaaggcc gg
1220812DNAHomo sapiens
208cgccggtgga at
1220912DNAHomo sapiens 209gaacccgtcc ta
1221012DNAHomo sapiens 210gattcatcag cg
1221112DNAHomo sapiens
211acaccggtct tc
1221212DNAHomo sapiens 212atcgtgccct aa
1221312DNAHomo sapiens 213gcgtcaatgt tc
1221412DNAHomo sapiens
214ctccgtatct cg
1221512DNAHomo sapiens 215ccgattcctt cg
1221612DNAHomo sapiens 216tgcgcctcca gt
1221712DNAHomo sapiens
217taacgtcgga gc
1221812DNAHomo sapiens 218aaggtcgccc at
1221912DNAHomo sapiens 219gtcggggact at
1222012DNAHomo sapiens
220ttcgagcgat tt
1222112DNAHomo sapiens 221tgagtcgtcg ag
1222212DNAHomo sapiens 222tttacgcaga gg
1222312DNAHomo sapiens
223aggaagtatc gc
1222412DNAHomo sapiens 224actcgatacc at
1222512DNAHomo sapiens 225cgctacatag ca
1222612DNAHomo sapiens
226ttcataaccg gc
1222712DNAHomo sapiens 227ccaaacggtt aa
1222812DNAHomo sapiens 228cgattccttc gt
1222912DNAHomo sapiens
229cgtcatgaat aa
1223012DNAHomo sapiens 230agtggcgatg ac
1223112DNAHomo sapiens 231cccctacggc ac
1223212DNAHomo sapiens
232gccaacccgc ac
1223312DNAHomo sapiens 233tgggacaccg gt
1223412DNAHomo sapiens 234ttgactgcgg cg
1223512DNAHomo sapiens
235actatgcgta gg
1223612DNAHomo sapiens 236tcacccaaag cg
1223712DNAHomo sapiens 237gcaggacgtc cg
1223812DNAHomo sapiens
238acaccgaaaa cg
1223912DNAHomo sapiens 239cggtgtattg ag
1224012DNAHomo sapiens 240cacgaggtat gc
1224112DNAHomo sapiens
241taaagcgacc cg
1224212DNAHomo sapiens 242cttagtcggc ca
1224312DNAHomo sapiens 243cgaaaacgtg gc
1224412DNAHomo sapiens
244cgtgccctga ac
1224512DNAHomo sapiens 245tttaccatcg aa
1224612DNAHomo sapiens 246cgtagccatg tt
1224712DNAHomo sapiens
247cccaaacggt ta
1224812DNAHomo sapiens 248gcgttatcag aa
1224912DNAHomo sapiens 249tcgatggtaa ac
1225012DNAHomo sapiens
250cgactttttg ca
1225112DNAHomo sapiens 251tcgacgactc ac
1225212DNAHomo sapiens 252acgcgtcaga ta
1225312DNAHomo sapiens
253cgtacggcac ag
1225412DNAHomo sapiens 254ctatgccgtg ca
1225512DNAHomo sapiens 255cgcgtcagat at
1225612DNAHomo sapiens
256aagatcggta gc
1225712DNAHomo sapiens 257cttcgcaagg ag
1225812DNAHomo sapiens 258gtcgtggact ac
1225912DNAHomo sapiens
259ggtcgtcatc aa
1226012DNAHomo sapiens 260gttaacagcg tg
1226112DNAHomo sapiens 261tagctaaccg tt
1226212DNAHomo sapiens
262agtaaaggcg ct
1226312DNAHomo sapiens 263ggtaatttcg tg
12264147RNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 264nnnnnnnnnn nnnnnnnnnn guuuuaguac ucuguaauuu uagguaugag
guagacgaaa 60auuguacuua uaccuaaaau uacagaaucu acuaaaacaa ggcaaaaugc
cguguuuauc 120ucgucaacuu guuggcgaga uuuuuuu
147
User Contributions:
Comment about this patent or add new information about this topic: