Crispr-cas Component Systems, Methods And Compositions For Sequence Manipulation

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.

Description

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application is a divisional of U.S. application Ser. No. 14/497,627 filed Sep. 26, 2014, which is a continuation in part of international patent application serial no. PCT/US2013/074611 filed Dec. 12, 2013 which claims priority to U.S. provisional patent applications 61/736,527, 61/748,427, 61/757,972, 61/768,959, 61/791,409 and 61/835,931 having Broad reference BI-2011/008/WSGR Docket No. 44063-701.101, BI-2011/008/WSGR Docket No. 44063-701.102, Broad reference BI-2011/008/VP Docket No. 44790.01.2003, BI-2011/008/VP Docket No. 44790.02.2003 and BI-2011/008/VP Docket No. 44790.03.2003 respectively, all entitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec. 12, 2012, Jan. 2, 2013, Jan. 29, 2013, Feb. 25, 2013, Mar. 15, 2013 and Jun. 17, 2013, respectively.

[0002] Reference is made to U.S. 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. Reference is also made to U.S. provisional patent applications 61/835,936, 61/836,127, 61/836,101, 61/836,080, 61/836,123 and 61/835,973 each filed Jun. 17, 2013. Reference is also made to U.S. provisional patent application 61/842,322 and U.S. patent application Ser. No. 14/054,414, each having Broad reference BI-2011/008A, entitled CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS filed on Jul. 2, 2013 and Oct. 15, 2013 respectively.

[0004] 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.

SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 31, 2015, is named 44790.14.2003_SL.txt and is 308,602 bytes in size.

BACKGROUND OF THE INVENTION

[0007] 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

[0008] 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.

[0009] 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 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%0, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. Determining optimal alignment is within the purview of one of skill in the art. For example, there are publically and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython and SeqMan. 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.

[0010] 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).

[0011] 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 Ill 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 .beta.-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1.alpha. 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 .beta.-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.).

[0012] Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells.

[0013] 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.

[0014] 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.

[0015] 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 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 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.

[0016] 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%/0 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.

[0017] 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.

[0018] 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.

[0019] 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 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.

[0020] 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 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.

[0021] 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.

[0022] 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.

[0023] 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. .sctn.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.

[0024] 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

[0025] 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:

[0026] 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) .about.3 bp upstream of the PAM (red triangle).

[0027] FIG. 2A-2F shows an exemplary CRISPR system and a possible mechanism of action (FIG. 2A), an example adaptation for expression in eukaryotic cells (FIG. 2B) and results of tests assessing nuclear localization and CRISPR activity (FIG. 2D). FIG. 2C discloses SEQ ID NOS 279-280, respectively, in order of appearance. FIG. 2E discloses SEQ ID NOS 281-283, respectively, in order of appearance, and FIG. 2F discloses SEQ ID NOS 284-288, respectively, in order of appearance.

[0028] FIG. 3A-3C show an exemplary expression cassette for expression of CRISPR system elements in eukaryotic cells (FIG. 3A), predicted structures of example guide sequences (FIG. 3B), and CRISPR system activity as measured in eukaryotic and prokaryotic cells (SEQ ID NOS 289-298, respectively, in order of appearance) (FIG. 3C).

[0029] FIG. 4A-4D shows results of an evaluation of SpCas9 specificity for an example target. FIG. 4A discloses SEQ ID NOS 299, 282 and 300-310, respectively, in order of appearance. FIG. 4C discloses SEQ ID NO: 299, 282 and 300-310, respectively, in order of appearance. FIG. 4C discloses SEQ ID NO: 299. FIGS. 4B and 4D show the results of the evaluation of SpCas9 specificity for the example target.

[0030] FIG. 5A-5G show an exemplary vector system (FIGS. 5A and 5C) and results for use in directing homologous recombination in eukaryotic cells (FIGS. 5B and 5D). FIG. 5E discloses SEQ ID NO: 311. FIG. 5F discloses SEQ ID NOS 312-313, respectively, in order of appearance. FIG. 5G discloses SEQ ID NOS 314-318, respectively, in order of appearance.

[0031] FIG. 6 provides a table of protospacer sequences (SEQ ID NOS 33, 32, 31, 322-327, 35, 34 and 330-334, respectively, in order of appearance) and summarizes modification efficiency results for protospacer targets designed based on exemplary S. pyogenes and S. thermophilus CRISPR systems with corresponding PAMs against loci in human and mouse genomes. Cells were transfected with Cas9 and either pre-crRNA/tracrRNA or chimeric RNA, and analyzed 72 hours after transfection. Percent indels are calculated based on Surveyor assay results from indicated cell lines (N=3 for all protospacer targets, errors are S.E.M., N.D. indicates not detectable using the Surveyor assay, and N.T. indicates not tested in this study).

[0032] FIG. 7A-7C shows a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting (FIGS. 7B-7C). FIG. 7A discloses SEQ ID NOS 335-336, respectively, in order of appearance.

[0033] FIG. 8 shows a schematic of a surveyor nuclease assay for detection of double strand break-induced micro-insertions and -deletions.

[0034] FIG. 9A-9B shows exemplary bicistronic expression vectors for expression of CRISPR system elements in eukaryotic cells. FIG. 9A discloses SEQ ID NOS 337-339, respectively, in order of appearance. FIG. 9B discloses SEQ ID NOS 344-342, respectively, in order of appearance.

[0035] FIG. 10A-10D shows a bacterial plasmid transformation interference assay (FIG. 10C), expression cassettes and plasmids (FIG. 10B) used therein, and transformation efficiencies of cells used therein (FIG. 10D). FIG. 10A discloses SEQ ID NOS 343-345, respectively, in order of appearance.

[0036] FIG. 11A-11C shows histograms of distances between adjacent S. pyogenes SF370 locus 1 PAM (NGG) (FIG. 11A) and S. thermophilus LMD9 locus 2 PAM (NNAGAAW) (FIG. 11B) in the human genome; and distances for each PAM by chromosome (Chr) (FIG. 1C).

[0037] FIG. 12A-12C shows an exemplary CRISPR system (FIG. 12A), an example adaptation for expression in eukaryotic cells (FIG. 12B), and results of tests assessing CRISPR activity. FIG. 12B discloses SEQ ID NOS 346-347, respectively, in order of appearance. FIG. 12C discloses SEQ ID NO: 348.

[0038] FIG. 13A-13C shows exemplary manipulations and results (FIG. 13C) of a CRISPR system for targeting of genomic loci in mammalian cells. FIG. 13A discloses SEQ ID NO: 349. FIG. 13B discloses SEQ ID NOS 350-352, respectively, in order of appearance.

[0039] FIG. 14A-14B shows the results of a Northern blot analysis of crRNA processing in mammalian cells (FIG. 14B). FIG. 14A discloses SEQ ID NO: 353.

[0040] FIG. 15A-15B shows an exemplary selection of protospacers in the human PVALB and mouse Th loci. FIG. 15A discloses SEQ ID NO: 354. FIG. 15B discloses SEQ ID NO: 355.

[0041] FIG. 16 shows example protospacer and corresponding PAM sequence targets of the S. thermophilus CRISPR system in the human EMX1 locus (SEQ ID NO: 348).

[0042] FIG. 17 provides a table of sequences for primers and probes (SEQ ID NOS 36-39 and 356-363, respectively, in order of appearance) used for Surveyor, RFLP, genomic sequencing, and Northern blot assays.

[0043] FIG. 18A-18C shows exemplary manipulation of a CRISPR system with chimeric RNAs and results of SURVEYOR assays for system activity in eukaryotic cells (FIGS. 18B and 18C). FIG. 18A discloses SEQ ID NO: 364.

[0044] FIG. 19A-19B shows two graphical representations of the results of SURVEYOR assays for CRISPR system activity in eukaryotic cells (SEQ ID NOS 365-443, respectively, in order of appearance).

[0045] FIG. 20 shows an exemplary visualization of some S. pyogenes Cas9 target sites in the human genome using the UCSC genome browser.

[0046] FIG. 21 shows predicted secondary structures for exemplary chimeric RNAs comprising a guide sequence, tracr mate sequence, and tracr sequence (SEQ ID NOS 444-463, respectively, in order of appearance).

[0047] FIG. 22 shows exemplary bicistronic expression vectors for expression of CRISPR system elements in eukaryotic cells (SEQ ID NOS 464 and 341-342, respectively, in order of appearance).

[0048] FIG. 23A-23B shows that Cas9 nuclease activity against endogenous targets may be exploited for genome editing. (a) Concept of genome editing using the CRISPR system. The CRISPR targeting construct directed cleavage of a chromosomal locus and was co-transformed with an editing template that recombined with the target to prevent cleavage. Kanamycin-resistant transformants that survived CRISPR attack contained modifications introduced by the editing template. tracr, trans-activating CRISPR RNA; aphA-3, kanamycin resistance gene. (b) Transformation of crR6M DNA in R6.sup.82325 cells with no editing template, the R6 wild-type srtA or the R6370.1 editing templates. Recombination of either R6 srtA or R6.sup.370.1 prevented cleavage by Cas9. Transformation efficiency was calculated as colony forming units (cfu) per .mu.g of crR6M DNA; the mean values with standard deviations from at least three independent experiments are shown. PCR analysis was performed on 8 clones in each transformation. "Un." indicates the unedited srtA locus of strain R6.sup.8232.5; "Ed." shows the editing template. R6.sup.8232.5 and R6.sup.370.1 targets are distinguished by restriction with EaeI.

[0049] FIG. 24A-24C shows analysis of PAM and seed sequences that eliminate Cas9 cleavage. (a) PCR products with randomized PAM sequences or randomized seed sequences were transformed in crR6 cells (SEQ ID NOS 465-469, respectively, in order of appearance). These cells expressed Cas9 loaded with a crRNA that targeted a chromosomal region of R6.sup.8232.5 cells (highlighted in pink) that is absent from the R6 genome. More than 2.times.105 chloramphenicol-resistant transformants, carrying inactive PAM or seed sequences, were combined for amplification and deep sequencing of the target region. (b) Relative proportion of number of reads after transformation of the random PAM constructs in crR6 cells (compared to number of reads in R6 transformants). The relative abundance for each 3-nucleotide PAM sequence is shown. Severely underrepresented sequences (NGG) are shown in red; partially underrepresented one in orange (NAG) (c) Relative proportion of number of reads after transformation of the random seed sequence constructs in crR6 cells (compared to number of reads in R6 transformants). The relative abundance of each nucleotide for each position of the first 20 nucleotides of the protospacer sequence is shown (SEQ ID NO: 470). High abundance indicates lack of cleavage by Cas9, i.e. a CRISPR inactivating mutation. The grey line shows the level of the WT sequence. The dotted line represents the level above which a mutation significantly disrupts cleavage (See section "Analysis of deep sequencing data" in Example 5)

[0050] FIG. 25A-25F shows introduction of single and multiple mutations using the CRISPR system in S. pneumoniae. (a) Nucleotide and amino acid sequences of the wild-type and edited (green nucleotides; underlined amino acid residues) bgaA. The protospacer, PAM and restriction sites are shown (SEQ ID NOS 471-475 and 472, respectively, in order of appearance). (b) Transformation efficiency of cells transformed with targeting constructs in the presence of an editing template or control. (c) PCR analysis for 8 transformants of each editing experiment followed by digestion with BtgZI (R.fwdarw.A) and TseI (NE.fwdarw.AA). Deletion of bgaA was revealed as a smaller PCR product. (d) Miller assay to measure the .beta.-galactosidase activity of WT and edited strains. (e) For a single-step, double deletion the targeting construct contained two spacers (in this case matching srtA and bgaA) and was co-transformed with two different editing templates (f) PCR analysis for 8 transformants to detect deletions in srtA and bgaA loci. 6/8 transformants contained deletions of both genes.

[0051] FIG. 26A-26D provides mechanisms underlying editing using the CRISPR system. (a) A stop codon was introduced in the erythromycin resistance gene ermAM to generate strain JEN53. The wild-type sequence can be restored by targeting the stop codon with the CRISPR::ermAM (stop) construct, and using the ermAM wild-type sequence as an editing template. (b) Mutant and wild-type ermAAM sequences (SEQ ID NOS 476-479, respectively, in order of appearance). (c) Fraction of erythromicyn-resistant (erm.sup.R) cfu calculated from total or kanamycin-resistant (kan.sup.R) cfu. (d) Fraction of total cells that acquire both the CRISPR construct and the editing template. Co-transformation of the CRISPR targeting construct produced more transformants (t-test, p=0.011). In all cases the values show the mean.+-.s.d. for three independent experiments.

[0052] FIG. 27A-27D illustrates genome editing with the CRISPR system in E. coli. (a) A kanamycin-resistant plasmid carrying the CRISPR array (pCRISPR) targeting the gene to edit may be transformed in the HME63 recombineering strain containing a chloramphenicol-resistant plasmid harboring cas9 and tracr (pCas9), together with an oligonucleotide specifying the mutation. (b) A K42T mutation conferring streptomycin resistance was introduced in the rpsL gene (SEQ ID NOS 480-483, respectively, in order of appearance) (c) Fraction of streptomicyn-resistant (strep.sup.R) cfu calculated from total or kanamycin-resistant (kan.sup.R) cfu. (d) Fraction of total cells that acquire both the pCRISPR plasmid and the editing oligonucleotide. Co-transformation of the pCRISPR targeting plasmid produced more transformants (t-test, p=0.00.sup.4). In all cases the values showed the mean.+-.s.d. for three independent experiments.

[0053] FIG. 28A-28B illustrates the transformation of crR6 genomic DNA leads to editing of the targeted locus (a) The IS1167 element of S. pneumoniae R6 was replaced by the CRISPR01 locus of S. pyogenes SF370 to generate crR6 strain. This locus encodes for the Cas9 nuclease, a CRISPR array with six spacers, the tracrRNA that is required for crRNA biogenesis and Cas1, Cas2 and Csn2, proteins not necessary for targeting. Strain crR6M contains a minimal functional CRISPR system without cas1, cas2 and csn2. The aphA-3 gene encodes kanamycin resistance. Protospacers from the streptococcal bacteriophages 8232.5 and 370.1 were fused to a chloramphenicol resistance gene (cat) and integrated in the srtA gene of strain R6 to generate strains R68232.5 and R6370.1. (b) Left panel: Transformation of crR6 and crR6M genomic DNA in R6.sup.8232.5 and R6370.1. As a control of cell competence a streptomycin resistant gene was also transformed. Right panel: PCR analysis of 8 R6.sup.8232.5 transformants with crR6 genomic DNA. Primers that amplify the srtA locus were used for PCR. 7/8 genotyped colonies replaced the R68232.5 srtA locus by the WT locus from the crR6 genomic DNA.

[0054] FIG. 29A-29F provides chromatograms of DNA sequences of edited cells obtained in this study. In all cases the wild-type and mutant protospacer and PAM sequences (or their reverse complement) are indicated. When relevant, the amino acid sequence encoded by the protospacer is provided. For each editing experiment, all strains for which PCR and restriction analysis corroborated the introduction of the desired modification were sequenced. A representative chromatogram is shown. (a) Chromatogram for the introduction of a PAM mutation into the R6.sup.8232.5 target (FIG. 23d) (SEQ ID NOS 484-485, respectively, in order of appearance). (b) Chromatograms for the introduction of the R>A and NE>AA mutations into .beta.-galactosidase (bgaA) (FIG. 25c) (SEQ ID NOS 471-475 and 472, respectively, in order of appearance). (c) Chromatogram for the introduction of a 6664 bp deletion within bgaA ORF (FIGS. 25c and 25f). The dotted line indicates the limits of the deletion (SEQ ID NOS 486-488, respectively, in order of appearance). (d) Chromatogram for the introduction of a 729 bp deletion within srtA ORF (FIG. 25f). The dotted line indicates the limits of the deletion (SEQ ID NOS 489-491, respectively, in order of appearance). (e) Chromatograms for the generation of a premature stop codon within ermAM (FIG. 33) (SEQ ID NOS 492-495, respectively, in order of appearance). (f) rpsL editing in E. coli (FIG. 27) (SEQ ID NOS 480-483, respectively, in order of appearance).

[0055] FIG. 30A-30C illustrates CRISPR immunity against random S. pneumoniae targets containing different PAMs. (a) Position of the 10 random targets on the S. pneumoniae R6 genome. The chosen targets have different PAMs and are on both strands. (b) Spacers corresponding to the targets were cloned in a minimal CRISPR array on plasmid pLZ12 and transformed into strain crR6Rc, which supplies the processing and targeting machinery in trans. (c) Transformation efficiency of the different plasmids in strain R6 and crR6Rc. No colonies were recovered for the transformation of pDB99-108 (T1-T10) in crR6Rc. The dashed line represents limit of detection of the assay.

[0056] FIG. 31 provides a general scheme for targeted genome editing. To facilitate targeted genome editing, crR6M was further engineered to contain tracrRNA, Cas9 and only one repeat of the CRISPR array followed by kanamycin resistance marker (aphA-3), generating strain crR6Rk. DNA from this strain is used as a template for PCR with primers designed to introduce a new spacer (green box designated with N). The left and right PCRs are assembled using the Gibson method to create the targeting construct. Both the targeting and editing constructs are then transformed into strain crR6Rc, which is a strain equivalent to crR6Rk but has the kanamycin resistance marker replaced by a chloramphenicol resistance marker (cat). About 90% of the kanamycin-resistant transformants contain the desired mutation.

[0057] FIG. 32 illustrates the distribution of distances between PAMs. NGG and CCN that are considered to be valid PAMs. Data is shown for the S. pneumoniae R6 genome as well as for a random sequence of the same length and with the same GC-content (39.7%). The dotted line represents the average distance (12) between PAMs in the R6 genome.

[0058] FIG. 33A-33D illustrates CRISPR-mediated editing of the ermAM locus using genomic DNA as targeting construct. To use genomic DNA as targeting construct it is necessary to avoid CRISPR autoimmunity, and therefore a spacer against a sequence not present in the chromosome must be used (in this case the ermAM erythromycin resistance gene). (a) Nucleotide and amino acid sequences of the wild-type and mutated (red letters) ermAM gene. The protospacer and PAM sequences are shown (SEQ ID NOS 492-495, respectively, in order of appearance). (b) A schematic for CRISPR-mediated editing of the ermAM locus using genomic DNA. A construct carrying an ermAM-targeting spacer (blue box) is made by PCR and Gibson assembly, and transformed into strain crR6Rc, generating strain JEN37. The genomic DNA of JEN37 was then used as a targeting construct, and was co-transformed with the editing template into JEN38, a strain in which the srtA gene was replaced by a wild-type copy of ermAM. Kanamycin-resistant transformants contain the edited genotype (JEN43). (c) Number of kanamycin-resistant cells obtained after co-transformation of targeting and editing or control templates. In the presence of the control template 5.4.times.10.sup.3; cfu/ml were obtained, and 4.3.times.10.sup.5 cfu/ml when the editing template was used. This difference indicates an editing efficiency of about 99% [(4.3.times.10.sup.5-5.4.times.10)/4.3.times.10]. (d) To check for the presence of edited cells seven kanamycin-resistant clones and JEN38 were streaked on agar plates with (erm+) or without (erm-) erythromycin. Only the positive control displayed resistance to erythromycin. The ermAM mut genotype of one of these transformants was also verified by DNA sequencing (FIG. 29e).

[0059] FIG. 34A-34D illustrates sequential introduction of mutations by CRISPR-mediated genome editing. (a) A schematic for sequential introduction of mutations by CRISPR-mediated genome editing. First, R6 is engineered to generate crR6Rk. crR6Rk is co-transformed with a srtA-targeting construct fused to cat for chloramphenicol selection of edited cells, along with an editing construct for a .DELTA.srtA in-frame deletion. Strain crR6 .DELTA.srtA is generated by selection on chlramphenicol. Subsequently, the .DELTA.srtA strain is co-transformed with a bgaA-targeting construct fused to aphA-3 for kanamycin selection of edited cells, and an editing construct containing a .DELTA.bgaA in-frame deletion. Finally, the engineered CRISPR locus can be erased from the chromosome by first co-transforming R6 DNA containing the wild-type IS1167 locus and a plasmid carrying a bgaA protospacer (pDB97), and selection on spectinomycin. (b) PCR analysis for 8 chloramphenicol (Cam)-resistant transformants to detect the deletion in the srtA locus. (c) .beta.-galactosidase activity as measured by Miller assay. In S. pneumoniae, this enzyme is anchored to the cell wall by sortase A. Deletion of the srtA gene results in the release of .beta.-galactosidase into the supernatant. .DELTA.bgaA mutants show no activity. (d) PCR analysis for 8 spectinomycin (Spec)-resistant transformants to detect the replacement of the CRISPR locus by wild-type IS1167.

[0060] FIG. 35A-35C illustrates the background mutation frequency of CRISPR in S. pneumoniae. (a) Transformation of the CRISPR::O or CRISPR::erm (stop) targeting constructs in JEN53, with or without the ermAM editing template. The difference in kan.sup.R CFU between CRISPR::O and CRISPR::erm (stop) indicates that Cas9 cleavage kills non-edited cells. Mutants that escape CRISPR interference in the absence of editing template are observed at a frequency of 3.times.10.sup.-3. (b) PCR analysis of the CRISPR locus of escapers shows that 7/8 have a spacer deletion. (c) Escaper #2 carries a point mutation in cas9 (SEQ ID NOS 496-499, respectively, in order of appearance).

[0061] FIG. 36 illustrates that the essential elements of the S. pyogenes CRISPR locus 1 are reconstituted in E. coli using pCas9. The plasmid contained tracrRNA, Cas9, as well as a leader sequence driving the crRNA array. The pCRISPR plasmids contained the leader and the array only. Spacers may be inserted into the crRNA array between BsaI sites using annealed oligonucleotides (SEQ ID NOS 343, 500 and 127, respectively, in order of appearance). Oligonucleotide design is shown at bottom. pCas9 carried chloramphenicol resistance (CmR) and is based on the low-copy pACYC184 plasmid backbone. pCRISPR is based on the high-copy number pZE21 plasmid. Two plasmids were required because a pCRISPR plasmid containing a spacer targeting the E. coli chromosome may not be constructed using this organism as a cloning host if Cas9 is also present (it will kill the host).

[0062] FIG. 37 illustrates CRISPR-directed editing in E. coli MG1655. An oligonucleotide (W542) carrying a point mutation that both confers streptomycin resistance and abolishes CRISPR immunity, together with a plasmid targeting rpsL (pCRISPR::rpsL) or a control plasmid (pCRISPR::O) were co-transformed into wild-type E. coli strain MG1655 containing pCas9. Transformants were selected on media containing either streptomycin or kanamycin. Dashed line indicates limit of detection of the transformation assay.

[0063] FIG. 38A-38B illustrates the background mutation frequency of CRISPR in E. coli HME63. (a) Transformation of the pCRISPR::O or pCRISPR::rpsL plasmids into HME63 competent cells. Mutants that escape CRISPR interference were observed at a frequency of 2.6.times.10.sup.-4. (b) Amplification of the CRISPR array of escapers showed that 8/8 have deleted the spacer.

[0064] FIG. 39A-39D shows a circular depiction of the phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (.about.1400 amino acids) and two of small Cas9s (.about.1100 amino acids).

[0065] FIG. 40A-40F shows the linear depiction of the phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (.about.1400 amino acids) and two of small Cas9s (.about.1100 amino acids).

[0066] FIG. 41A-41M shows sequences where the mutation points are located within the SpCas9 gene (SEQ ID NOS 501-502, respectively, in order of appearance).

[0067] FIG. 42 shows a schematic construct in which the transcriptional activation domain (VP64) is fused to Cas9 with two mutations in the catalytic domains (D10 and H840).

[0068] FIG. 43A-43D shows genome editing via homologous recombination. (a) Schematic of SpCas9 nickase, with D10A mutation in the RuvC 1 catalytic domain. (b) Schematic representing homologous recombination (HR) at the human EMX1 locus using either sense or antisense single stranded oligonucleotides as repair templates. Red arrow above indicates sgRNA cleavage site; PCR primers for genotyping (Tables J and K) are indicated as arrows in right panel. (c) Sequence of region modified by HR. d, SURVEYOR assay for wildtype (wt) and nickase (D10A) SpCas9-mediated indels at the EMX1 target 1 locus (n=3) (SEQ ID NOS 503-505, 503, 506 and 505, respectively, in order of appearance). Arrows indicate positions of expected fragment sizes.

[0069] FIG. 44A-44B shows single vector designs for SpCas9. FIG. 44A discloses SEQ ID NOS 320-321 and 328, respectively, in order of appearance. FIG. 44B discloses SEQ ID NO: 329.

[0070] FIG. 45 shows quantification of cleavage of NLS-Csn1 constructs NLS-Csn1, Csn1, Csn1-NLS, NLS-Csn1-NLS, NLS-Csn1-GFP-NLS and UnTFN.

[0071] FIG. 46 shows index frequency of NLS-Cas9, Cas9, Cas9-NLS and NLS-Cas9-NLS.

[0072] FIG. 47 shows a gel demonstrating that SpCas9 with nickase mutations (individually) do not induce double strand breaks.

[0073] FIG. 48A-48B shows a design of the oligo DNA used as Homologous Recombination (HR) template in this experiment (FIG. 48A) and a comparison of HR efficiency induced by different combinations of Cas9 protein and HR template (FIG. 48B).

[0074] FIG. 49A shows the Conditional Cas9, Rosa26 targeting vector map.

[0075] FIG. 49B shows the Constitutive Cas9, Rosa26 targeting vector map.

[0076] FIG. 50A-50H show the sequences of each element present in the vector maps of FIGS. 49A-B (SEQ ID NOS 507-516, respectively, in order of appearance).

[0077] FIG. 51 shows a schematic of the important elements in the Constitutive and Conditional Cas9 constructs.

[0078] FIG. 52 shows the functional validation of the expression of Constitutive and Conditional Cas9 constructs.

[0079] FIG. 53 shows the validation of Cas9 nuclease activity by Surveyor.

[0080] FIG. 54 shows the quantification of Cas9 nuclease activity.

[0081] FIG. 55 shows construct design and homologous recombination (HR) strategy.

[0082] FIG. 56 shows the genomic PCR genotyping results for the constitutive (Right) and conditional (Left) constructs at two different gel exposure times (top row for 3 min and bottom row for 1 min).

[0083] FIG. 57 shows Cas9 activation in mESCs.

[0084] FIG. 58 shows a schematic of the strategy used to mediate gene knockout via NHEJ using a nickase version of Cas9 along with two guide RNAs.

[0085] FIG. 59 shows how DNA double-strand break (DSB) repair promotes gene editing. In the error-prone non-homologous end joining (NHEJ) pathway, the ends of a DSB are processed by endogenous DNA repair machineries and rejoined together, which can result in random insertion/deletion (indel) mutations at the site of junction. Indel mutations occurring within the coding region of a gene can result in frame-shift and a premature stop codon, leading to gene knockout. Alternatively, a repair template in the form of a plasmid or single-stranded oligodeoxynucleotides (ssODN) can be supplied to leverage the homology-directed repair (HDR) pathway, which allows high fidelity and precise editing.

[0086] FIG. 60 shows the timeline and overview of experiments. Steps for reagent design, construction, validation, and cell line expansion. Custom sgRNAs (light blue bars) for each target, as well as genotyping primers, are designed in silico via our online design tool (available at the website genome-engineering.org/tools). sgRNA expression vectors are then cloned into a plasmid containing Cas9 (PX330) and verified via DNA sequencing. Completed plasmids (pCRISPRs), and optional repair templates for facilitating homology directed repair, are then transfected into cells and assayed for ability to mediate targeted cleavage. Finally, transfected cells can be clonally expanded to derive isogenic cell lines with defined mutations.

[0087] FIG. 61A-61C shows Target selection and reagent preparation. (a) For S. pyogenes Cas9, 20-bp targets (highlighted in blue) must be followed by 5'-NGG, which can occur in either strand on genomic DNA. We recommend using the online tool described in this protocol in aiding target selection (www.genome-engineering.org/tools). (b) Schematic for co-transfection of Cas9 expression plasmid (PX165) and PCR-amplified U6-driven sgRNA expression cassette. Using a U6 promoter-containing PCR template and a fixed forward primer (U6 Fwd), sgRNA-encoding DNA can appended onto the U6 reverse primer (U6 Rev) and synthesized as an extended DNA oligo (Ultramer oligos from IDT). Note the guide sequence (blue N's) in U6 Rev is the reverse complement of the 5'-NGG flanking target sequence (SEQ ID NOS 517 and 517-519, respectively, in order of appearance). (c) Schematic for scarless cloning of the guide sequence oligos into a plasmid containing Cas9 and sgRNA scaffold (PX330). The guide oligos (blue N's) contain overhangs for ligation into the pair of BbsI sites on PS330, with the top and bottom strand orientations matching those of the genomic target (i.e. top oligo is the 20-bp sequence preceding 5'-NGG in genomic DNA). Digestion of PX330 with BbsI allows the replacement of the Type IIs restriction sites (blue outline) with direct insertion of annealed oligos. It is worth noting that an extra G was placed before the first base of the guide sequence. Applicants have found that an extra G in front of the guide sequence does not adversely affect targeting efficiency. In cases when the 20-nt guide sequence of choice does not begin with guanine, the extra guanine will ensure the sgRNA is efficiently transcribed by the U6 promoter, which prefers a guanine in the first base of the transcript (SEQ ID NOS 320-321 and 328, respectively, in order of appearance).

[0088] FIG. 62A-62D shows the anticipated results for multiplex NHEJ. (a) Schematic of the SURVEYOR assay used to determine indel percentage. First, genomic DNA from the heterogeneous population of Cas9-targeted cells is amplified by PCR. Amplicons are then reannealed slowly to generate heteroduplexes. The reannealed heteroduplexes are cleaved by SURVEYOR nuclease, whereas homoduplexes are left intact. Cas9-mediated cleavage efficiency (% indel) is calculated based on the fraction of cleaved DNA, as determined by integrated intensity of gel bands. (b) Two sgRNAs (orange and blue bars) are designed to target the human GRIN2B and DYRK1A loci. SURVEYOR gel shows modification at both loci in transfected cells. Colored arrows indicated expected fragment sizes for each locus. (c) A pair of sgRNAs (light blue and green bars) are designed to excise an exon (dark blue) in the human EMX1 locus. Target sequences and PAMs (red) are shown in respective colors, and sites of cleavage indicated by red triangle. Predicted junction is shown below. Individual clones isolated from cell populations transfected with sgRNA 3, 4, or both are assayed by PCR (OUT Fwd, OUT Rev), reflecting a deletion of .about.270-bp. Representative clones with no modification (12/23), mono-allelic (10/23), and bi-allelic (1/23) modifications are shown. IN Fwd and IN Rev primers are used to screen for inversion events (FIG. 6d) (SEQ ID NOS 520-522, respectively, in order of appearance). (d) Quantification of clonal lines with EMX1 exon deletions. Two pairs of sgRNAs (3.1, 3.2 left-flanking sgRNAs; 4.1, 4.2, right flanking sgRNAs) are used to mediate deletions of variable sizes around one EMX1 exon. Transfected cells are clonally isolated and expanded for genotyping analysis for deletions and inversion events. Of the 105 clones are screened, 51 (49%) and 11 (10%) carrying heterozygous and homozygous deletions, respectively. Approximate deletion sizes are given since junctions may be variable.

[0089] FIG. 63A-63C shows the application of ssODNs and targeting vectors (FIG. 63A) to mediate HR with both wildtype and nickase mutant of Cas9 in HEK293FT and HUES9 cells with efficiencies ranging from 1.0-27% (FIG. 63C). FIG. 63B discloses SEQ ID NOS 503-505, 503, 506 and 505, respectively, in order of appearance.

[0090] FIG. 64 shows a schematic of a PCR-based method for rapid and efficient CRISPR targeting in mammalian cells. A plasmid containing the human RNA polymerase III promoter U6 is PCR-amplified using a U6-specific forward primer and a reverse primer carrying the reverse complement of part of the U6 promoter, the sgRNA (+85) scaffold with guide sequence, and 7 T nucleotides for transcriptional termination. The resulting PCR product is purified and co-delivered with a plasmid carrying Cas9 driven by the CBh promoter (SEQ ID NOS 517, 523, 518 and 524-525, respectively, in order of appearance).

[0091] FIG. 65 shows SURVEYOR Mutation Detection Kit from Transgenomics results for each gRNA and respective controls. A positive SURVEYOR result is one large band corresponding to the genomic PCR and two smaller bands that are the product of the SURVEYOR nuclease making a double-strand break at the site of a mutation. Each gRNA was validated in the mouse cell line, Neuro-N2a, by liposomal transient co-transfection with hSpCas9. 72 hours post-transfection genomic DNA was purified using QuickExtract DNA from Epicentre. PCR was performed to amplify the locus of interest.

[0092] FIG. 66 shows Surveyor results for 38 live pups (lanes 1-38) 1 dead pup (lane 39) and I wild-type pup for comparison (lane 40). Pups 1-19 were injected with gRNA Chd8.2 and pups 20-38 were injected with gRNA Chd8.3. Of the 38 live pups, 13 were positive for a mutation. The one dead pup also had a mutation. There was no mutation detected in the wild-type sample. Genomic PCR sequencing was consistent with the SURVEYOR assay findings (SEQ ID NOS 526-528, respectively, in order of appearance).

[0093] FIG. 67 shows a design of different Cas9 NLS constructs. All Cas9 were the human-codon-optimized version of the Sp Cas9. NLS sequences are linked to the cas9 gene at either N-terminus or C-terminus. All Cas9 variants with different NLS designs were cloned into a backbone vector containing so it is driven by EF1.alpha. promoter. On the same vector there is a chimeric RNA targeting human EMX1 locus driven by U6 promoter, together forming a two-component system.

[0094] FIG. 68 shows the efficiency of genomic cleavage induced by Cas9 variants bearing different NLS designs. The percentage indicate the portion of human EMX1 genomic DNA that were cleaved by each construct. All experiments are from 3 biological replicates. n=3, error indicates S.E.M.

[0095] FIG. 69A shows a design of the CRISPR-TF (Transcription Factor) with transcriptional activation activity. The chimeric RNA is expressed by U6 promoter, while a human-codon-optimized, double-mutant version of the Cas9 protein (hSpCas9m), operably linked to triple NLS and a VP64 functional domain is expressed by a EF1.alpha. promoter. The double mutations, DIOA and H840A, renders the cas9 protein unable to introduce any cleavage but maintained its capacity to bind to target DNA when guided by the chimeric RNA.

[0096] FIG. 69B shows transcriptional activation of the human SOX2 gene with CRISPR-TF system (Chimeric RNA and the Cas9-NLS-VP64 fusion protein). 293FT cells were transfected with plasmids bearing two components: (1) U6-driven different chimeric RNAs targeting 20-bp sequences within or around the human SOX2 genomic locus, and (2) EF1a-driven hSpCas9m (double mutant)-NLS-VP64 fusion protein. 96 hours post transfection, 293FT cells were harvested and the level of activation is measured by the induction of mRNA expression using a qRT-PCR assay. All expression levels are normalized against the control group (grey bar), which represents results from cells transfected with the CRISPR-TF backbone plasmid without chimeric RNA. The qRT-PCR probes used for detecting the SOX2 mRNA is Taqman Human Gene Expression Assay (Life Technologies). All experiments represents data from 3 biological replicates, n=3, error bars show s.e.m.

[0097] FIG. 70 depicts NLS architecture optimization for SpCas9.

[0098] FIG. 71 shows a QQ plot for NGGNN sequences.

[0099] FIG. 72 shows a histogram of the data density with fitted normal distribution (black line) and 0.99 quantile (dotted line).

[0100] FIG. 73A-73C shows RNA-guided repression of bgaA expression by dgRNA::cas9**. a. The Cas9 protein binds to the tracrRNA, and to the precursor CRISPR RNA which is processed by RNAseIII to form the crRNA. The crRNA directs binding of Cas9 to the bgaA promoter and represses transcription. b. The targets used to direct Cas9** to the bgaA promoter are represented (SEQ ID NO: 529). Putative-35, -10 as well as the bgaA start codon are in bold. c. Betagalactosidase activity as measure by Miller assay in the absence of targeting and for the four different targets.

[0101] FIG. 74A-74E shows characterization of Cas9** mediated repression. a. The gfpmut2 gene and its promoter, including the -35 and -10 signals are represented together with the position of the different target sites used the study. b. Relative fluorescence upon targeting of the coding strand. c. Relative fluorescence upon targeting of the non-coding strand. d. Northern blot with probes B477 and B478 on RNA extracted from T5, T10, B10 or a control strain without a target. e. Effect of an increased number of mutations in the 5' end of the crRNA of B1, T5 and B10.

[0102] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

[0103] 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.

[0104] 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)".

[0105] 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.

[0106] 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.

[0107] 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.

[0108] "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.

[0109] 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 1, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, N.Y.

[0110] "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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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)).

[0118] 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.

[0119] 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.

[0120] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET lid (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0121] In some embodiments, a vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (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.).

[0122] 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).

[0123] 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.

[0124] 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 a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

[0125] 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 Mycobacterium 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, Myvcobacterium, Streptomyces. Aquifex. Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrcoccus, Campylobacter, Wolinella, Acinetobacter. Erwinia, Escherichia. Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

[0126] 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 hybridization 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 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.

[0127] 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 hybridize 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%0, 70%, 80.degree. %., 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.

[0128] 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.

[0129] 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, Cas1B, 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 1 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.

[0130] As a further example, two or more catalytic domains of Cas9 (RuvC 1, 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.

[0131] 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, P A), 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.

[0132] 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 (SEQ ID NO: 1); the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 2)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4); the hRNPAI M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 5); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 9) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 10) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 11) and PKQKKRK (SEQ ID NO: 12) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 13) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 14) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 16) of the steroid hormone receptors (human) glucocorticoid.

[0133] 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.

[0134] 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%0/6, 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.

[0135] 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 (SEQ ID NO: 530) where NNNNNNNNNNNNXGG (SEQ ID NO: 531) (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 (SEQ ID NO: 532) where NNNNNNNNNNNXGG (SEQ ID NO: 533) (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 (SEQ ID NO: 17) where NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 18) (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 MMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 19) where NNNNNNNNNNNXXAGAAW (SEQ ID NO: 20) (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 MMMMMMMMXGGXG (SEQ ID NO: 534) where NNNNNNNNNNNNXGGXG (SEQ ID NO: 535) (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 (SEQ ID NO: 536) where NNNNNNNNNNNXGGXG (SEQ ID NO: 537) (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.

[0136] 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 (attorney docket 44790.11.2022; Broad Reference BI-2013/004A); incorporated herein by reference.

[0137] 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) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa- ggctt catgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTT (SEQ ID NO: 21); (2) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaa- atca acaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 22); (3) NNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatca acaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 23); (4) NNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaa agtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 24); (5) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttg- aa aaagtgTTTTTTT (SEQ ID NO: 25); and (6) gNNNNNNNNtNNNNNNNNNNNtttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTT TTT (SEQ ID NO: 26). 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).

[0138] 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.

[0139] 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.

[0140] 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).

[0141] 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.TM. and Lipofectin.sup.TM). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, 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).

[0142] 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).

[0143] 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.

[0144] 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).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).

[0145] 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 w.sup.2 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 be 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.

[0146] 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, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, 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/ARI, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells. Ku812, KCL22, KGI, KYOl, 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-5F, 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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 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 15). Accordingly, reference herein to animal cells may also apply, mutatis mutandis, to plant cells unless otherwise apparent.

[0151] 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).

[0152] 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.

[0153] 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 be provide individually or in combinations, and may be 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.

[0154] 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.

[0155] 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.

[0156] 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 recognized 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.

[0157] 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 U.S. provisional patent applications 61/736,527 and 61/748,427 having Broad reference BI-2011/008/WSGR Docket No. 44063-701.101 and 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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 U.S. Provisional application 61/736,527 filed on Dec. 12, 2012 and 61/748,427 filed on Feb. 2, 2013. Such genes, proteins and pathways may be the target polynucleotide of a CRISPR complex.

TABLE-US-00001 TABLE A DISEASE/ DISORDERS 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; Igf1 (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 Aber; Ccl2; Cc2; cp (ceruloplasmin); Timp3; cathepsinD; Macular Vldlr; Ccr2 Degeneration Schizophrenia Neuregulin1 (Nrg1); Erb4 (receptor for Neuregulin); Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2 Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b Disorders 5-HTT (Slc6a4); COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1) Trinucleotide HTT (Huntington's Dx); SBMA/SMAX1/AR (Kennedy's Repeat Dx); FXN/X25 (Friedrich's Ataxia); ATX3 (Machado- Disorders 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 Fragile X FMR2; FXR1; FXR2; mGLUR5 Syndrome Secretase APH-1 (alpha and beta); Presenilin (Psen1); nicastrin Related (Ncstn); .PEN-2 Disorders Others Nos1: Parp1; Nat1; Nat2 Prion--related Prp disorders ALS SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c) Drug Prkce (alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2; addiction Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol) Autism Mecp2; BZRAP1; MDGA2; Sema5A.; Neurexin 1; Fragile X (FMR2 (AFF2); FXR1; FXR2; Mglur5) Alzheimer's E1; CHIP; UCH; UBB; Tau; LRP; PICALM; Clusterin; Disease PS1; SORL1; CR1; Vldlr; Ubal; Uba3; CHIP28 (Aqp1, Aquaporin 1); Uchl1; Uchl3; APP Inflammation IL-10; IL-1 (IL-1a; IL-1b); IL-13; IL-17 (IL-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 x-Synuclein; DJ-1; LRRK2; Parkin; PINK1 Disease

TABLE-US-00002 TABLE B Blood and Anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, coagulation diseases PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, and 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, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCT; 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, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia (HBA2, HBB, HBD, LCRB, HBA1) Cell dysregulation B-cell non-Hodgkin lymphoma. (BCL7A, BCL7); Leukemia (TAL1, and oncology TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, diseases and disorders HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, 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 and AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, immune related SDF1); Autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, diseases and disorders FAS, CD95, ALPS1A); Combined immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); 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), II-23, Cx3cr1, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1); Severe combined immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4). Metabolic, liver. Amyloid neuropathy (TTR, PALB); Amyloidosis APOA1, APP, AAA, kidney and protein CVAP, AD1, GSN, FGA, LYZ, TTR, PALB); Cirrhosis (KRT18, KRT8, diseases and disorders CIRH1A, NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7, CF, MRP7); Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GRE1, 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/Skeletal Becker muscular dystrophy (DMD, BMD, MYF6), Duchenne Muscular diseases and disorders Dystrophy (DMD, BMD); Emery-Dreifuss muscular dystrophy (LMNA, LMN1, 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, SGCA, ADL, DAG2, LGMD2D, 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, CATF I, SMARD1). Neurological and ALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, neuronal diseases and VEGF-c); Alzheimer disease (APP, AAA, CVAP, AD1, APOE, AD2, disorders PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1, 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, NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16, 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 (Slc6a4), COMT, DRD (Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1)); Secretase Related Disorders (APH-1 (alpha and beta), Presenilin (Psen1), 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 diseases and Age-related macular degeneration (Abcr, Ccl2, Cc2, cp (ceruloplasmin), disorders Timp3, cathepsinD, Vldlr, Ccr2); Cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PTI X3, .13FSP2, CP49, CP47, (RYAN., CRY A1, 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, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1); Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma (MYOC, TIGR, GLC1A, 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, RP20, 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 Signaling PRKCE; ITGAM; ITGA5; IRAK1; PRKAA2; EIF2AK2; 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; CDKN1A; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK ; PDPK1; PPP2R5C; CTNNB1 ; MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN; ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP9OAA1; RPS6KB1 ERK/MAPK Signaling PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2; RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; 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; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3; ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAT; ATF4; PRKCA; SRF; STAT1; SGK Glucocorticoid Receptor RAC1; TAF4B; EP300; SMAD2; TRAF6; PCAF; ELK1; Signaling MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA; CREB1; FOS; HSPA5; NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8; BCL2L1; MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A; MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3; MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP CDKN1A; MAP2K2; JAK1; IL8; NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1; SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 Axonal Guidance PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; Signaling IGF1; RAC1; RAP1A; EIF4E; 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 Receptor PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1; AKT2; DOK1; 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 Cytoskeleton ACTN4; PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; INS; ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1; PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN; VIL2; RAF1; GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1; PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGK Huntington's Disease PRKCE; IGF1; EP300; RCOR1; 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 Signaling PRKCE; ROCK1; BID; IRAK1; PRKAA2; EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2L1; CAPN1; MAPK3; CASP8; KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG; RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA; CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 B Cell Receptor RAC1; PTEN; LYN; 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 Extravasation ACTN4; CD44; PRKCE; 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 Signaling ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1; ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3; MAPK8; CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7; PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1; TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3 Acute Phase Response IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11; Signaling AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8; RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1; TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3; IL1R1; IL6 PTEN Signaling ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11; MAPK1; 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; BRCA1; GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53LNP1; BCL2; PIK3CB; PIK3C3; MAPK8; THBS1; ATR; BCL2L1; E2F1 PMAIP1; CHEK2; TNFRSF10B; TP73; RB1; HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1; RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2; GSK3B; BAX; AKT3 Aryl Hydrocarbon HSPB1; EP300; FASN; TGM2; RXRA; MAPK1; NQO1; Receptor NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; Signaling 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; IL6; CYP1B1; HSP90AA1 Xenobiotic Metabolism PRKCE; EP300; PRKCZ; 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; PIK3C2A; PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; .MAP2K1; NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1 SAPK/JNK Signaling PRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2; PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1; IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3; CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK PPAr/RXR Signaling PRKAA2; EP300; INS; 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; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1; ADIPOQ NF-KB Signaling IRAK1; EIF2AK2; EP300; INS; MYD88; PRKCZ; TRAF6; TBK1; 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 Signaling ERBB4; PRKCE; ITGAM; ITGA5; PTEN; PRKCZ; ELK1; MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3; ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17; AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC; NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt & Beta catenin CD44; EP300; LRP6; 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 Receptor PTEN; INS; EIF4E; 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 IL-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1; MAPK1; 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; IL1R1; SRF; IL6 Hepatic Cholestasis PRKCE; IRAK1; INS; MYD88; 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; NFKB1; ESR1; SREBF1; FGFR4; JUN; IL1R1; PRKCA; IL6 IGF-1 Signaling IGF1; PRKCZ; ELK1; MAPK1; PTPN11; NEDD4; AKT2; PIK3CA; PRKCZ; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R; IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; ICFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF; CTGF; RPS6KB1 NRF2-mediated PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1; Oxidative NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8; Stress Response PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A; MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN; KEAP1 GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic Fibrosis/Hepatic EDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; Stellate Cell Activation SMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9 IGF1R; IL6R; RELA; TLR4; PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1; CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 PPAR Signaling EP300; INS; TRAF6; PPARA; RXRA; MAPK1; IKBKB; 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 RI Signaling PRKCE; RAC1; PRKCZ; LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK; MAPK14; TNF; RAF1; FYN MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; VANT3; PRKCA G-Protein Coupled PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB; Receptor Signaling PIK3CA; CREB1; GNAQ; NEKB2; CAMK2A; PIK3CB; PIK3C3; MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; NK11; PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCA Inositol Phosphate PRKCE; IRAK1; PRKAA2; 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 Signaling EIF2AK2; ELK1; ABL2; 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 Signaling ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA; ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA; AKT3; FOXO1; PRKCA Natural Killer Cell PRKCE; RAC1; PRKCZ; MAPK1; RAC2; PTPN11; Signaling KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; PRKD1; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4; AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: G1/S HDAC4; SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; Checkpoint Regulation ATR; ABL1; E2F1; HDAC2; FIDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1; HDAC6 T Cell Receptor RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS; Signaling NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA; PIK3C2A; BTK; LCK; RAF1; IKBKG; RELB; FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK; BCL10; JUN; VAV3 Death Receptor Signaling CRADD; HSPB1; BID; BIRC4; TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX; TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1; CASP2; BIRC2; CASP3; BIRC3 FGF Signaling RAC1; FGFR1; MET; MAPKAPK2; MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3; MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1; FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF GM-CSF Signaling LYN; ELK1; MAPK1; 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 Lateral BID; IGF1; RAC1; BIRC4; PGF; CAPNS1; CAPN2; Sclerosis Signaling PIK3CA; BCL2; PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/Stat Signaling PTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS; SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1 AKT1; JAK2; PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1 Nicotinate and PRKCE; IRAK1; PRKAA2; EIF2AK2; GRK6; MAPK1; Nicotinamide PLK1; AKT2; CDK8; MAPK8; MAPK3; PRKCD; PRKAA1; Metabolism PBEF1; MAPK9; CDK2; PIM1; DYRK1A; MAP2K2; MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine Signaling CXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8; MAPK3; KRAS; MAPK13; RHOA; CCR3; SRC; PPP1CC ; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1; JUN; CCL2; PRKCA IL-2 Signaling ELK1; MAPK1; PTPN11; AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3 Synaptic Long Term PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1; GNAS; Depression PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCA Estrogen Receptor TAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2; Signaling SMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 Protein Ubiquitination TRAF6; SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; Pathway CBL; UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1; VHL; USP90AA1; 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/RXR Activation PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKCI; CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1; LRP5; CEBPB; FOXO1; PRKCA TGF-beta Signaling EP300; SMAD2; SMURF1; MAPK1; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1; RAF1; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5 Toll-like Receptor IRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; Signaling IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN p38 MAPK Signaling HSPB1; IRAK1; TRAF6; MAPKAPK2; ELK1; FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1 Neurotrophin/TRK NTRK2; MAPK1; PTPN11; PIK3CA; CREB1; FOS; Signaling PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A; RAF1; .MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42; JUN; ATF4 FXR/RXR Activation INS; PPARA; FASN; RXRA; AKT2; SDC1; MAPK8; APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; FOXO1 Synaptic Long Term PRKCE; RAP1A; EP300; PRKCZ; MAPK1; CREB1; Potentiation PRKCI; GNAQ; CAMK2A; PRKD1; MAPK3; KRAS; PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4; PRKCA Calcium Signaling RAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1; CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4; HDAC6 EGF Signaling ELK1; MAPK1; EGFR; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1 Hypoxia Signaling in the EDN1; PTEN; EP300; NQO1; UBE2I; CREB1; ARNT; Cardiovascular System HIF1A; SLC2A4; NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN; ATF4; VHL; HSP90AA1 LPS/IL-1 Mediated IRAK1; MYD88; TRAF6; PPARA; RXRA; ABCA1; Inhibition of RXR MAPK8; ALDH1A1; GSTP1; MAPK9; ABCB1; TRAF2; Function TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN; IL1R1 LXR/RXR Activation FASN; RXRA; NCOR2; ABCA1; NFKB2; IRF3; RELA; NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1; IL1R1; CCL2; IL6; MMP9 Amyloid Processing PRKCE; CSNK1E; MAPK1; CAPNS1; AKT2; CAPN2; CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1; GSK3B; AKT3; APP IL-4 Signaling AKT2; PIK3CA; PIK3CB; PIK3C3; RS1; KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1; AKT3; RPS6KB1 Cell Cycle: G2/M DNA EP300; PCAF; BRCA1; GADD45A; PLK1; BTRC; Damage Checkpoint CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A; Regulation PRKDC; ATM; SFN; CDKN2A Nitric Oxide Signaling KDR; FLT1; PGF; AKT2; PIK3CA; PIK3CB; PIK3C3; in the CAV1; PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1; Cardiovascular System VEGFA; AKT3; HSP90AA1 Purine Metabolism NME2; SMARCA4; MYH9; RRM2; ADAR; EIF2AK4; PKM2; ENTPD1; RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; NME1 cAMP-mediated RAP1A; MAPK1; GNAS; CREB1; CAMK2A; MAPK3; Signaling SRC; RAF1; MAP2K2; STAT3; MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8; CASP8; .MAPK10; MAPK9; CASP9; Dysfunction PARK7; PSEN1; PARK2; APP; CASP3 Notch Signaling HES1; JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3; NOTCH1; DLL4 Endoplasmic Reticulum HSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4; Stress Pathway EIF2AK3; CASP3 Pyrimidine Metabolism NME2; AICDA; RRM2; EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson's Signaling UCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7; RARK2; CASP3 Cardiac & Beta GNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; Adrenergic Signaling PPP2R5C Glycolysis/ HK2; GCK; GPI; ALDH1A1; PKM2; LDHA; HK1 Gluconeogenesis Interferon Signaling IRF1; SOCS1; JAK1; JAK2; IFITM1; STAT1; IFIT3 Sonic Hedgehog ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B; DYRK1B Signaling Glycerophospholipid PLD1; GRN; GPAM; YWHAZ; SPHK1; SPHK2 Metabolism Phospholipid PRDX6; PLD1; GRN; YWHAZ; SPFIK1; SPHK2 Degradation Tryptophan Metabolism SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1; SIAH1 Lysine Degradation SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C Nucleotide Excision ERCC5; ERCC4; XPA; XPC; ERCC1 Repair Pathway Starch and Sucrose UCHL1; HK2; GCK; GPI; HK1 Metabolism Aminosugars Metabolism NQO1; HK2; GCK; HK1 Arachidonic Acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism Circadian Rhythm CSNK1E; CREB1; ATF4; NR1D1 Signaling Coagulation System BDKRB1; F2R; SERPINE1; F3 Dopamine Receptor PPP2R1A; PPP2CA; PPP1CC; PPP2R5C Signaling Glutathione Metabolism IDH2; GSTP1; ANPEP; IDH1 Glycerolipid Metabolism ALDH1A1; GPAM; SPHK1; SPHK2 Linoleic Acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism Methionine Metabolism DNMT1; DNMT3B; AHCY; DNMT3A Pyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA Arginine and Proline ALDH1A1; NOS3; NOS2A Metabolism Eicosanoid Signaling PRDX6; GRN; YWHAZ Fructose and Mannose HK2; GCK; HK1 Metabolism Galactose Metabolism HK2; GCK; HK1 Stilbene, Coumarine and PRDX6; PRDX1; 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; Cnr1; cnr2; Grk2; Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5; Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial Function AIF; CytC; SMAC (Diablo); Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd); Noggin (Nog); WNT (Wnt2; Neurology Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b; Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin; Dkk-1; Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1; unc-86 (Pou4f1 or Brn3a); Numb; Reln

[0162] 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. McIvor 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.

[0163] 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).

[0164] In yet another aspect of the invention, the CRISPR-Cas system may be 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.

[0165] 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 (website at health.nih.gov/topic/GeneticDisorders). 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.

[0166] 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 and 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.

[0167] Examples of proteins associated with Parkinson's disease include but are not limited to .alpha.-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1, Synphilin-1, and NURR1.

[0168] Examples of addiction-related proteins may include ABAT for example.

[0169] Examples of inflammation-related proteins may include the monocyte chemoattractant protein-1 (MCP 1) 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.

[0170] Examples of cardiovascular diseases associated proteins may include IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin) synthase), MB (myoglobin), 1L4 (interleukin 4), ANGPT 1I (angiopoietin 1), ABCG8 (ATP-binding cassette, sub-family G (WHITE), member 8), or CTSK (cathepsin K), for example.

[0171] 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 (UBEIC) encoded by the UBA3 gene, for example.

[0172] Examples of proteins associated 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.

[0173] Examples of proteins associated 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.

[0174] Examples of proteins associated Schizophrenia may include NRG1, ErbB4, CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, and combinations thereof.

[0175] 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.

[0176] 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), APHIB (anterior pharynx defective I homolog B (C. elegans)), PSEN2 (presenilin 2 (Alzheimer disease 4)), or BACE1 (beta-site APP-cleaving enzyme 1), for example.

[0177] Examples of proteins associated with Amyotrophic Lateral Sclerosis may include SODI (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.

[0178] Examples of proteins associated with prion diseases may include SODI (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.

[0179] 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 ADRAID (Alpha-1D adrenergic receptor for Alpha-1D adrenoreceptor), for example.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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); Alstrom 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 [COFSI]; 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 II; 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.

[0185] 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

[0186] 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

[0187] An example type II CRISPR system is the type II 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.

[0188] Cell Culture and Transfection

[0189] Human embryonic kidney (HEK) cell line HEK 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), 100 U/mL penicillin, and 100 .mu.g/mL streptomycin at 37.degree. C. with 5.degree. % CO.sub.2 incubation. Mouse neuro2A (N2A) cell line (ATCC) was maintained with DMEM supplemented with 5% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100 U/mL penicillin, and 100 .mu.g/mL streptomycin at 37.degree. C. with 5% CO.sub.2.

[0190] HEK 293FT or N2A cells were seeded into 24-well plates (Corning) one day prior to transfection at a density of 200,000 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 800 ng of plasmids were used.

[0191] Surveyor Assay and Sequencing Analysis for Genome Modification

[0192] HEK 293FT or N2A cells were transfected with plasmid DNA as described above.

[0193] After transfection, the cells were incubated at 37.degree. C. for 72 hours before genomic DNA extraction. Genomic DNA was extracted using the QuickExtract DNA extraction kit (Epicentre) following the manufacturer's protocol. Briefly, cells were resuspended in QuickExtract solution and incubated at 65.degree. C. for 15 minutes and 98.degree. C. for 10 minutes. Extracted genomic DNA was immediately processed or stored at -20.degree. C.

[0194] The genomic region surrounding a CRISPR target site for each gene was PCR amplified, and products were purified using QiaQuick Spin Column (Qiagen) following manufacturer's protocol. A total of 400 ng of the purified PCR products were mixed with 2 .mu.l 10.times.Taq polymerase PCR buffer (Enzymatics) and ultrapure water to a final volume of 20 .mu.l, and subjected to a re-annealing process to enable heteroduplex formation: 95.degree. C. for 10 min, 95.degree. C. to 85.degree. C. ramping at -2.degree. C./s, 85.degree. C. to 25.degree. C. at -0.25.degree. C./s, and 25.degree. 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, as a measure of the fraction of cleaved DNA. FIG. 8 provides a schematic illustration of this Surveyor assay.

[0195] Restriction fragment length polymorphism assay for detection of homologous recombination

[0196] HEK 293FT and N2A cells were transfected with plasmid DNA, and incubated at 37.degree. C. for 72 hours before genomic DNA extraction as described above. The target genomic region was PCR amplified using primers outside the homology arms of the homologous recombination (HR) template. PCR products were separated on a 1% agarose gel and extracted with MinElute GelExtraction Kit (Qiagen). Purified products were digested with HindIII (Fermentas) and analyzed on a 6% Novex TBE poly-acrylamide gel (Life Technologies).

[0197] RNA Secondary Structure Prediction and Analysis

[0198] RNA secondary structure prediction was performed using 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).

[0199] Bacterial Plasmid Transformation Interference Assay

[0200] Elements of the S. pyogenes CRISPR locus 1 sufficient for CRISPR activity were reconstituted in E. coli using pCRISPR plasmid (schematically illustrated in FIG. 10A). pCRISPR contained tracrRNA, SpCas9, and a leader sequence driving the crRNA array. Spacers (also referred to as "guide sequences") were inserted into the crRNA array between BsaI sites using annealed oligonucleotides, as illustrated. Challenge plasmids used in the interference assay were constructed by inserting the protospacer (also referred to as a "target sequence") sequence along with an adjacent CRISPR motif sequence (PAM) into pUC19 (see FIG. 10B). The challenge plasmid contained ampicillin resistance. FIG. 10C provides a schematic representation of the interference assay. Chemically competent E. coli strains already carrying pCRISPR and the appropriate spacer were transformed with the challenge plasmid containing the corresponding protospacer-PAM sequence. pUC19 was used to assess the transformation efficiency of each pCRISPR-carrying competent strain. CRISPR activity resulted in cleavage of the pPSP plasmid carrying the protospacer, precluding ampicillin resistance otherwise conferred by pUC19 lacking the protospacer. FIG. 10D illustrates competence of each pCRISPR-carrying E. coli strain used in assays illustrated in FIG. 4C.

[0201] RNA Purification

[0202] HEK 293FT cells were maintained and transfected as stated above. Cells were harvested by trypsinization followed by washing in phosphate buffered saline (PBS). Total cell RNA was extracted with TRI reagent (Sigma) following manufacturer's protocol. Extracted total RNA was quantified using Naonodrop (Thermo Scientific) and normalized to same concentration.

[0203] Northern Blot Analysis of crRNA and tracrRNA Expression in Mammalian Cells

[0204] RNAs were mixed with equal volumes of 2X loading buffer (Ambion), heated to 95.degree. C. for 5 min, chilled on ice for 1 min, and then loaded onto 8% denaturing polyacrylamide gels (SequaGel, National Diagnostics) after pre-running the gel for at least 30 minutes. The samples were electrophoresed for 1.5 hours at 40 W limit. Afterwards, the RNA was transferred to Hybond N+ membrane (GE Healthcare) at 300 mA in a semi-dry transfer apparatus (Bio-rad) at room temperature for 1.5 hours. The RNA was crosslinked to the membrane using autocrosslink button on Stratagene UV Crosslinker the Stratalinker (Stratagene). The membrane was pre-hybridized in ULTRAhyb-Oligo Hybridization Buffer (Ambion) for 30 min with rotation at 42.degree. C., and probes were then added and hybridized overnight. Probes were ordered from IDT and labeled with [gamma-.sup.32P]ATP (Perkin Elmer) with T4 polynucleotide kinase (New England Biolabs). The membrane was washed once with pre-warmed (42.degree. C.) 2.times.SSC, 0.5% SDS for 1 min followed by two 30 minute washes at 42.degree. C. The membrane was exposed to a phosphor screen for one hour or overnight at room temperature and then scanned with a phosphorimager (Typhoon).

[0205] Bacterial CRISPR System Construction and Evaluation

[0206] CRISPR locus elements, including tracrRNA, Cas9, and leader were PCR amplified from Streptococcus pyogenes SF370 genomic DNA with flanking homology arms for Gibson Assembly. Two BsaI type IIS sites were introduced in between two direct repeats to facilitate easy insertion of spacers (FIG. 9). PCR products were cloned into EcoRV-digested pACYC184 downstream of the tet promoter using Gibson Assembly Master Mix (NEB). Other endogenous CRISPR system elements were omitted, with the exception of the last 50 bp of Csn2. Oligos (Integrated DNA Technology) encoding spacers with complimentary overhangs were cloned into the BsaI-digested vector pDC000 (NEB) and then ligated with T7 ligase (Enzymatics) to generate pCRISPR plasmids. Challenge plasmids containing spacers with PAM sequences (also referred to herein as "CRISPR motif sequences") were created by ligating hybridized oligos carrying compatible overhangs (Integrated DNA Technology) into BamHI-digested pUC19. Cloning for all constructs was performed in E. co/i strain JM109 (Zymo Research).

[0207] pCRISPR-carrying cells were made competent using the Z-Competent E. coli Transformation Kit and Buffer Set (Zymo Research, T3001) according to manufacturer's instructions. In the transformation assay, 50 uL aliquots of competent cells carrying pCRISPR were thawed on ice and transformed with 1 ng of spacer plasmid or pUC19 on ice for 30 minutes, followed by 45 second heat shock at 42.degree. C. and 2 minutes on ice. Subsequently, 250 ul SOC (Invitrogen) was added followed by shaking incubation at 37.degree. C. for lhr, and 100 uL of the post-SOC outgrowth was plated onto double selection plates (12.5 ug/ml chloramphenicol, 100 ug/ml ampicillin). To obtain cfu/ng of DNA, total colony numbers were multiplied by 3.

[0208] 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 (2.times.NLS-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 (SEQ ID NO: 2)), and the C-terminal NLS was that of the SV40 large T-antigen (PKKKRKV (SEQ ID NO: 1)). Of the versions of SpCas9 tested, only 2.times.NLS-SpCas9 exhibited nuclear localization (FIG. 2B).

[0209] 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 171 nt that are subsequently processed into identical 75 nt mature tracrRNAs. The shorter 89 nt tracrRNA was selected for expression in mammalian cells (expression constructs illustrated in FIG. 7A, with functionality as determined by results of the Surveyor assay shown in FIG. 7B). 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 (.about.75 bp). Very low amounts of long tracrRNA are detected on the Northern blot.

[0210] 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 EMY1 locus (FIG. 2C), a key gene in the development of the cerebral cortex.

[0211] 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 (FIG. 8) (see e.g. Guschin el 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. 4-7, 12, and 13). These results define a three-component system for efficient CRISPR-mediated genome modification in mammalian cells. 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 EMX1 genomic locus (FIG. 7B).

[0212] FIG. 14 provides an additional Northern blot analysis of crRNA processing in mammalian cells. FIG. 14A 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 (see FIG. 6) and the direct repeat sequences are shown in the sequence beneath FIG. 14A. The line indicates the region whose reverse-complement sequence was used to generate Northern blot probes for EMX1(1) crRNA detection. FIG. 14B 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 .about.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.

[0213] 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 EF1.alpha. promoter and tracrRNA and pre-crRNA array (DR-Spacer-DR) driven by the RNA Po13 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 .mu.m.

[0214] 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). To increase co-delivery efficiency, a bicistronic expression vector was created to drive co-expression of a chimeric RNA and SpCas9 in transfected cells (FIGS. 3A and 8). In parallel, the bicistronic vectors were used to express a pre-crRNA (DR-guide sequence-DR) with SpCas9, to induce processing into crRNA with a separately expressed tracrRNA (compare FIG. 13B top and bottom). FIG. 9 provides schematic illustrations of bicistronic expression vectors for pre-crRNA array (FIG. 9A) or chimeric crRNA (represented by the short line downstream of the guide sequence insertion site and upstream of the EF1.alpha. promoter in FIG. 9B) with hSpCas9, showing location of various elements and the point of guide sequence insertion. The expanded sequence around the location of the guide sequence insertion site in FIG. 9B also shows a partial DR sequence (GTTTTAGAGCTA (SEQ ID NO: 27)) and a partial tracrRNA sequence (TAGCAAGTTAAAATAAGGCTAGTCCGTTTTT (SEQ ID NO: 28)). Guide sequences can be inserted between BbsI sites using annealed oligonucleotides. Sequence design for the oligonucleotides are shown below the schematic illustrations in FIG. 9, with appropriate ligation adapters indicated. WPRE represents the Woodchuck hepatitis virus post-transcriptional regulatory element. The efficiency of chimeric RNA-mediated cleavage was tested by targeting the same EMX1 locus described above. Using both Surveyor assay and Sanger sequencing of amplicons, Applicants confirmed that the chimeric RNA design facilitates cleavage of human EMX1 locus with approximately a 4.7% modification rate (FIG. 4).

[0215] Generalizability of CRISPR-mediated cleavage in eukaryotic cells was tested by targeting additional genomic loci in both human and mouse cells by designing chimeric RNA targeting multiple sites in the human EMX1 and PVALB, as well as the mouse Th loci. FIG. 15 illustrates the selection of some additional targeted protospacers in human PVALB (FIG. 15A) and mouse Th (FIG. 15B) loci. Schematics of the gene loci and the location of three protospacers within the last exon of each are provided. The underlined sequences include 30 bp of protospacer sequence and 3 bp at the 3' end corresponding to the PAM sequences. 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 (FIGS. 3B and 6). 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 (FIG. 6).

[0216] FIG. 13 provides a further illustration that SpCas9 can be reprogrammed to target multiple genomic loci in mammalian cells. FIG. 13A provides a schematic of the human EMX1 locus showing the location of five protospacers, indicated by the underlined sequences. FIG. 13B 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. 13C. Each protospacer is targeted using either processed pre-crRNA/tracrRNA complex (crRNA) or chimeric RNA (chiRNA).

[0217] 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.

[0218] 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. 44, which illustrates 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. The vector shown in FIG. 44b includes a tracrRNA coding sequence linked to an H1 promoter.

[0219] 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. 10). 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. 10C). In the bacterial assay, all spacers facilitated efficient CRISPR interference (FIG. 4C). These results suggest that there may be additional factors affecting the efficiency of CRISPR activity in mammalian cells.

[0220] 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 EMX1, and FIG. 4D shows a Surveyor gel comparing the efficiency of TALEN and Cas9 (n=3).

[0221] 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 1 catalytic domain of SpCas9 was engineered to convert the nuclease into a nickase (SpCas9n; illustrated in FIG. 5A) (see e.g. Sapranauskas et al., 2011, Nucleic Acids 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.

[0222] 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. 4G) was detected. This demonstrates that the CRISPR system can mediate multiplexed editing within a single genome.

Example 2

CRISPR System Modifications and Alternatives

[0223] 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 Mg.sup.2+ 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 (FIG. 11, evaluating both plus and minus strands of human chromosomal sequences). 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. For example, FIG. 12 illustrates adaptation of the Type II CRISPR system from CRISPR 1 of Streptococcus thermophilus LMD-9 for heterologous expression in mammalian cells to achieve CRISPR-mediated genome editing. FIG. 12A provides a Schematic illustration of CRISPR 1 from S. thermophilus LMD-9. FIG. 12B illustrates the design of an expression system for the S. thermophilus CRISPR system. Human codon-optimized hStCa9 is expressed using a constitutive EF1.alpha. promoter. Mature versions of tracrRNA and crRNA are expressed using the U6 promoter to promote precise transcription initiation. Sequences from the mature crRNA and tracrRNA are illustrated. A single base indicated by the lower case "a" in the crRNA sequence is used to remove the polyU sequence, which serves as a RNA polIII transcriptional terminator. FIG. 12C provides a schematic showing guide sequences targeting the human EMX1 locus as well as their predicted secondary structures. 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, which is indicated by a rainbow scale reproduced in FIG. 12C in gray scale. FIG. 12D shows the results of hStCas9-mediated cleavage in the target locus using the Surveyor assay. 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. 6. FIG. 16 provides a schematic of additional protospacer and corresponding PAM sequence targets of the S. thermophilus CRISPR system in the human EMX1 locus. Two protospacer sequences are highlighted and their corresponding PAM sequences satisfying NNAGAAW motif are indicated by underlining 3' with respect to the corresponding highlighted sequence. Both protospacers target the anti-sense strand.

Example 3

Sample Target Sequence Selection Algorithm

[0224] 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'-N.sub.x-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'-N-NNAGAAW-3' (SEQ ID NO: 29) 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 N.sub.x may be fixed by the program or specified by the user, such as 20.

[0225] 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).

[0226] Further details of methods and algorithms to optimize sequence selection can be found in U.S. application Ser. No. 61/836,080 (attorney docket 44790.11.2022); incorporated herein by reference.

Example 4

Evaluation of Multiple Chimeric crRNA-tracrRNA Hybrids

[0227] 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 (SEQ ID NO: 30) 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. 19a and 19b, corresponding to FIGS. 18b and 18c, 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 proto- protospacer spacer genomic sequence ID target (5' to 3') PAM strand 1 EMX1 GGACATCGATGTCACCTCCAATG TGG + ACTAGGG (SEQ ID NO: 31) 2 EMX1 CATTGGAGGTGACATCGATGTCC TGG - TCCCCAT (SEQ ID NO: 32) 3 EMX1 GGAAGGGCCTGAGTCCGAGCAGA GGG + AGAAGAA (SEQ ID NO: 33) 4 PVALB GGTGGCGAGAGGGGCCGAGATTG AGG + GGTGTTC (SEQ ID NO: 34) 5 PVALB ATGCAGGAGGGTGGCGAGAGGGG TGG + CCGAGAT (SEQ ID NO: 35)

[0228] Cell Culture and Transfection

[0229] 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), 100 U/mL penicillin, and 100 g/mL streptomycin at 37.degree. C. with 5% CO.sub.2 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.

[0230] SURVEYOR Assay for Genome Modification

[0231] 293FT cells were transfected with plasmid DNA as described above. Cells were incubated at 37.degree. 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.degree. C. for 15 minutes and 98.degree. 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 .mu.l 10.times.Taq DNA Polymerase PCR buffer (Enzymatics) and ultrapure water to a final volume of 20 .mu.l, and subjected to a re-annealing process to enable heteroduplex formation: 95.degree. C. for 10 min, 95C to 85.degree. C. ramping at -2.degree. C./s, 85.degree. C. to 25.degree. C. at -0.25.degree. C./s, and 25.degree. 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 primer genomic primer sequence name target (5' to 3') Sp-EMX1-F EMX1 AAAACCACCCTTCTCTCTGGC (SEQ ID NO: 36) Sp-EMX1-R EMX1 GGAGATTGGAGACACGGAGAG (SEQ ID NO: 37) Sp-PVALB-F PVALB CTGGAAAGCCAATGCCTGAC (SEQ ID NO: 38) Sp-PVALB-R PVALB GGCAGCAAACTCCTTGTCCT (SEQ ID NO: 39)

[0232] Computational Identification of Unique CRISPR Target Sites

[0233] 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'-N.sub.20-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. 21.

[0234] 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. 18b and 19a). 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. 18c and 19b).

[0235] 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 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.

[0236] FIG. 22 illustrates an exemplary bicistronic expression vector 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 (tract: mate sequence CIFFITAGAGCTA (SEQ ID NO: 27), 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".

[0237] Sequences desctibed in the above examples are as follows (polynucleotide sequences are 5' to 3'):

TABLE-US-00006 U6-short tracrRNA (Streptococcus pyogenes SF370): (SEQ ID NO: 40) GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG CTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA CACCGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCG TTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (bold = tracrRNA sequence; underline = terminator sequence) U6-long tracrRNA (Streptococcus pyogenes SF370): (SEQ ID NO: 41) GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG CTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA CACCGGTAGTATTAAGTATTGTTTTATGGCTGATAAATTTCTTTGAATT TCTCCTTGATTATTTGTTATAAAAGTTATAAAATAATCTTGTTGGAACC ATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGCTTTTTTT U6-DR-BbsI backbone-DR (Streptococcus pyogenes SF370): (SEQ ID NO: 42) GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG CTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA CACCGGGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACGGGTCTT CGAGAAGACGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC U6-chimeric RNA-BbsI backbone (Streptococcus pyogenes SF370) (SEQ ID NO: 43) GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG CTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA CACCGGGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAA AATAAGGCTAGTCCG NLS-SpCas9-EGFP: (SEQ ID NO: 44) MDYKDUDGDYKDMDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLD IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QSKNGYAGIDGGASQEEFYKFIKPILEKNlDGTEELLVKLNREDIXRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQL IHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD ELVKVMGRHKPENIVIEMARENQTTOKGQKNSRERMKRIEEGIKELGSQ ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI TQRKFDNLTKAERGGLSELDKAGF1KRQLVETRQITKHVAQILDSRMNI KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT VAYSVLVVAKVEKGKSKKIASVKELLGITIMERSSFFKNPIDFLEAKGY KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFL YLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKIIRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDR KRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAAVSKGEELFTGV VPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL VTTLTYGVQCFSRYPDHMKOHDFFKSAMPEGYVQERTIFFKDDGNYKTR AEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQK NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSAL SKDPNEKRDHMVLLEFVTAAGITLGMDELYK SpCas9-EGFP-NLS: (SEQ ID NO. 45) MDKKYSIGLDIGTNSVGVVAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQHIFSNEMAKVDDSF FFIRLEESFLVEHDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDS IDKADLRLIYLALAHMIKFRGFIFLIEGDLNPDNSDVDKLFIQLVQTYN QLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFL AAKNLSDAILLSDILRVNTEITKAPLSASMfKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEEL LVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS AQSFIERMTNFDKNLPNEKVLPKIISLLYEYFTVYNELTKVKYVTEGMR KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVE DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIE ERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSP AKKGILQTVKVVDELVKVVIGRIIKPENIVIEMARENQTTQKGQKNSRE RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK KMKNYWRQLLNAKIJTQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQ ITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMER SSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDII EQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG APAAFKYFDTTTDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD AAAVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI CTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQER TIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY NSHNVYIMADKQKNGIKVTSTKIRHNIEDGSVQLADHYQQNTPIGDGPV LLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGFFLGMDELYKKRPA ATKKAGQAKKKK NLS-SpCas9-EGFP-NLS: (SEQ ID NO: 46) MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGTHGWAADKKYSIGLDI GTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTQKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYMQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLK IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILK EHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSF

LKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQR KFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKWFYSNMN FFKTEITLANGEIRKRPLIETNGETGEIVDKGRDFATVRKVLSMPQVNI VKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSV LVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGIIKRMLASAGELOKGNELALPSKYVNFLYLAS HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK VLSAYNKHRDKPIREOAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDAAAVSKGEELFTGVVPILV ELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVTWPTLVTTLT YGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKF EGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN EKRDHMVLLEFVTAAGITLGMDELYKKRPAATKKAGQAKKKK NLS-SpCas9-NLS: (SEQ ID NO: 47) MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLD IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA LARVIIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLS DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNPILEKIEKILTFRI PYWGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFD KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDK VMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIAMAGSPAIKKGILQTVKVVD ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP QSFLKDDSIDNKVLTRSDKNRGKSDNWSEEVVKKMKNYWRQLLNAKLIT QRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTK YDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLLARKKDVVDPKKYGGFDSP TVAYSVLVVAKVEKGKSKKLKSVKELLGITLMERSSFEKNPIDFLEAKG YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDR KRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKK KK NLS-mCherry-SpRNase3: (SEQ ID NO: 48) MFLFLSLTSFLSSSRTLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFE IEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPA DIPDYLKLSFPEGFKWERVMNTEDGGVVTVTQDSSLQDGEFIYKVKLRG TNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDA EVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRFIST GGMDELYKGSKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNV SHNERLEFLGDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAG FSRFCSFDAYIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAV RRFLKQVMIPQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGP AHAKQFEVSIVVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEV SpRNase3-mCherry-NLS: (SEQ ID NO: 49) MKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVSHNERLEFL GDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGFSRPCSFDA YIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVRRFLKQVMI PQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPAHAKQFEVS IVVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEVGSVSKGEEDNMAIIK EFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAW DILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTV TQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGA LKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVMKLDITSHNE DYTIVEQYERAEGRHSTGGMDELYKKRPAATKKAGQAKKKK NLS-SpCas9n-NLS (the D10A nickase mutation is lowercase): (SEQ ID NO: 50) MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLa IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERFIPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLS DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQIPFKYKEIFF DQSKNGYAGYIDGGASQEEFYKFDCPILEKMDGTEFXLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFD KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLITCTMIKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDD KVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKW DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGKELGSQ ILKEHPVENTQLQNEKLYLYYLQNGRDNlYVDQELDINRLSDYDVDHIV PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL ITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDS PTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASFFYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILA DANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAK KKK hEMX1-HR Template-HindII-NheI: (SEQ ID NO: 51) GAATGCTGCCCTCAGACCCGCTTCCTCCCTGTCCTTGTCTGTCCAAGGA GAATGAGGTCTCACTGGTGGATTTCGGACTACCCTGAGGAGCTGGCACC TGAGGGACAAGGCCCCCCACCTGCCCAGCTCCAGCCTCTGATGAGGGGT GGGAGAGAGCTACATGAGGTTGCTAAGAAAGCCTCCCCTGAAGGAGACC ACACAGTGTGTGAGGTTGGAGTCTCTAGCAGCGGGTTCTGTGCCCCCAG GGATAGTCTGGCTGTCCAGGCACTGCTCTTGATATAAACACCACCTCCT AGTTATGAAACCATGCCCATTCTGCCTCTCTGTATGGAAAAGAGCATGG GGCTGGCCCGTGGGGTGGTGTCCACTTTAGGCCCTGTGGGAGATCATGG GAACCCACGCAGTGGGTCATAGGCTCTCTCATTTACTACTCACATCCAC TCTGTGAAGAAGCGATTATGATCTCTCCTCTAGAAACTCGTAGAGTCCC ATGTCTGCCGGCTTCCAGAGCCTGCACTCCTCCACCTTGGCTTGGCTTT GCTGGGGCTAGAGGAGCTAGGATGCACAGCAGCTCTGTGACCCTTTGTT TGAGAGGAACAGGAAAACCACCCTTCTCTCTGGCCCACTGTGTCCTCTT CCTGCCCTGCCATCCCCTTCTGTGAATGTTAGACCCATGGGAGCAGCTG GTCAGAGGGGACCCCGGCCTGGGGCCCCTAACCCTATGTAGCCTCAGTC TTCCCATCAGGCTCTCAGCTCAGCCTGAGTGTTGAGGCCCCAGTGGCTG CTCTGGGGGCCTCCTGAGTTTCTCATCTGTGCCCCTCCCTCCCTGGCCC AGGTGAAGGTGTGGTTCCAGAACCGGAGGACAAAGTACAAACGGCAGAA GCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCAT CACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACA TCGATGTCACCTCCAATGACaagcttgctagcGGTGGGCAACCACAAAC CCACGAGGGCAGAGTGCTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCC CAAGCTGGACTCTGGCCACTCCCTGGCCAGGCTTTGGGGAGGCCTGGAG TCATGGCCCCACAGGGCTTGAAGCCCGGGGCCGCCATTGACAGAGGGAC

AAGCAATGGGCTGGCTGAGGCCTGGGACCACTTGGCCTTCTCCTCGGAG AGCCTGCCTGCCTGGGCGGGCCCGCCCGCCACCGCAGCCTCCCAGCTGC TCTCCGTGTCTCCAATCTCCCTTTTGTTTTGATGCATTTCTGTTTTAAT TTATTTTCCAGGCACCACTGTAGTTTAGTGATCCCCAGTGTCCCCCTTC CCTATGGGAATAATAAAAGTCTCTCTCTTAATGACACGGGCATCCAGCT CCAGCCCCAGAGCCTGGGGTGGTAGATTCCGGCTCTGAGGGCCAGTGGG GGCTGGTAGAGCAAACGCGTTCAGGGCCTGGGAGCCTGGGGTGGGGTAC TGGTGGAGGGGGTCAAGGGTAATTCATTAACTCCTCTCTTTTGTTGGGG GACCCTGGTCTCTACCTCCAGCTCCACAGCAGGAGAAACAGGCTAGACA TAGGGAAGGGCCATCCTGTATCTTGAGGGAGGACAGGCCCAGGTCTTTC TTAACGTATTGAGAGGTGGGAATCAGGCCCAGGTAGTTCAATGGGAGAG GGAGAGTGCTTCCCTCTGCCTAGAGACTCTGGTGGCTTCTCCAGTTGAG GAGAAACCAGAGGAAAGGGGAGGATTGGGGTCTGGGGGAGGGAACACCA TTCACAAAGGCTGACGGTTCCAGTCCGAAGTCGTGGGCCCACCAGGATG CTCACCTGTCCTTGGAGAACCGCTGGGCAGGTTGAGACTGCAGAGACAG GGCTTAAGGCTGAGCCTGCAACCAGTCCCCAGTGACTCAGGGCCTCCTC AGCCCAAGAAAGAGCAACGTGCCAGGGCCCGCTGAGCTCTTGTGTTCAC CTG NLS-StCsn1-NLS: (SEQ ID NO 52) MKRPAATKKAGQAKKKKSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSR IFPAAQAENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKI SINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSS VGDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRL INVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGP GNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEF NLLNDLNNLTWTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLS CDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDREILDKLAY VLTLNTEREGIQEALEHEFADGSFSOKQVDELVQFRKANSSIFGKGWHN FSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTE EIYNPWAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQK IQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGE RCLYTGKTISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQ EKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISK FDVRKKFIERNLVDTRYASRWLNALQEHFRAHKIDTKVSVVRGQFTSQL RRHWGTEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLD IETGELISDDEYKESVTKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNR KISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDK SKFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHG YIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNMCVVLQSVSPWRA DWFNKTTGKYEILGLKYADLQFEKGTGTYKISQEKYNDIKKKEGVDSDS EFKFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKF EGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGD KPKLDFKRPAATKKAGQAKKKK U6-St_tracrRNA(7-97): (SEQ ID NO: 53) GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGG CTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATT AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTT GAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAA CACCGTTACTTAAATCTTGCAGAAGCTACAAAGATAAGGCTTCATGCCG AAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTAA U6-DR-spacer-DR (S. pyogenes SF370) (SEQ ID NO: 54) gagggcctatttcccatgattccttcatatttgcatatacgatacaagg ctgttagagagataattggaattaatttgactgtaaacacaaagatatt agtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgca gttttaaaattatgttttaaaatggactatcatatgcttaccgtaactt gaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaa caccgggttttagagctatgctgttttgaatggtcccaaaacNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNgttttagagctatgctgttttgaatg gtcccaaaacTTTTTTT (lowercase underline = direct repeat; N = guide sequence; bold = terminator) Chimeric RNA containing +48 tracr RNA (S. pyogenes SF370) (SEQ ID NO: 55) gagggcctatttcccatgattccttcatatttgcatatacgatacaagg ctgttagagagataattggaattaatttgactgtaaacacaaagatatt agtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgca gttttaaaattatgttttaaaatggactatcatatgcttaccgtaactt gaaagtatttcgatttcaggctttatatatcttgtggaaaggacgaaac accNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagtta aaataaggctagtccgTTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Chimeric RNA containing +54 tracr RNA (S. pyogenes SF370) (SEQ ID NO: 56) gagggcctatttcccatgattccttcatatttgcatatacgatacaagg ctgttagagagataattggaattaatttgactgtaaacacaaagatatt agtacaaaatacgtgacgtagaaagtaataatttatgggtagtagcagt tttaaaattatgattaaaatggactatcatatgcttaccgtaacttgaa agtatttgatttcttggctttatatatcttgtggaaaggacgaaacacc NNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaa taaggctagtccgttatcaTTTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Chimeric RNA containing +67 tracr RNA (S. pyogenes SF370) (SEQ ID NO: 57) gagggcctatttcccatgattccttcatatttgcatatacgatacaagg ctgttagagagataattggaattaatttgactgtaaacacaaagatatt agtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgca gttttaaaattatgattaaaatggactatcatatgcttaccgtaacttg aaagtatttcgatttcaggctttatatatcttgtggaaaggacgaaaca ccNNNNNNNNNNNNNNNNNNNNgtttagagctagaaatagcaagttaaa ataaggctagtccgttatcaacttgaaaaagtaTTTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Chimeric RNA containing +85 tracr RNA (S. pyogenes SF370) (SEQ ID NO: 58) gagggcctatacccatgattccttcatatttgcatatacgatacaaggc tgttagagagataattggaattaatttgactgtaaacacaaagatatta gtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcag ttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttg aaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaac accNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagtta aaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtg cTTTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) CBh-NLS-SpCas9-NLS (SEQ ID NO: 59) CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAA TAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC TATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATC TCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTAT TTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGG GGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCG GCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGC GGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGT CGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCG CCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGG CGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAG GGATTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGA GCACCTGCCTGAAATCACTTTTTTTCAGGTTGGaccaataccaccATGG ACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAA AGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATC CACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCG GCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCC CAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAG AAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGG

CCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAA CCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTG GACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGG ATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGT GGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTG GTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGG CCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAA CCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACC TACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACG CCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAA TCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAAC CTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCG ACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGA CGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTG TTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCC TGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGAT CAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTC GTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGA GCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGA GTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAG GAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGA CCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCA CGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAAC CGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGG GCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAG CGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGC GCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACC TGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTT CACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATG AGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACC TGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGA CTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTG GAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAA TTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCT GGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATC GAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGA AGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAA GCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGAT TTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCC ACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTC CGGCCAGGGCGATAGCCTGGACGAGCACATTGCCAATCTGGCCGGCAGC CCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGC TCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAAT GGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAG AGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCC TGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTA CCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTG GACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGA GCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGA CAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAG AAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCC AGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGA ACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAG ATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGT ACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAA GTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTG CGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCG TCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTT CGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAG AGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCA ACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGAT CCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTG TGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGC CCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAG CAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGA AAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCG TGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAA GAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGA AGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACA AAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTT CGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTG CAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGT ACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGA GCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATC ATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTA ATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCAT CAGAGAGCAGCKrCGAGAATATCATCCACCTGTTTACCCTGACCAATCT GGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAG AGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGA GCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGG CGACTTTCTTTTTCTTAGCTTGACCAGCTTTCTTAGTAGCAGCAGGACG CTTTAA (underline = NLS-hSpCas9-NLS) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 21) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaa atcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccct gtcattttatggcagggtgttttacgttatttaaTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 22) NNNNNNNNNNNNNNNNNNNNgtattgtactctcaGAAAtgcagaagcta caaaataacttcatgccgaaatcaacaccctgtcattttatggcagggt gttacgttatttaaTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 23) NNNNNNNNNNNNNNNNNNNNgttttttgtactacaGAAAtgcagaagct acaaagataaggcttcatgccgaaatcaacaccctgtcattttatggca gggtgtTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 60) NNNNNNNNNNNNNNNNNNNNgttattgtactctcaagatttaGAAAtaa atcttgcagaagctacaaagataaggatcatgccgaaatcaacaccctg tcatatatggcagggtgttttcgttatttaaTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 61) NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagct acaaagataaggcttcatgccgaaatcaacaccctgtcattttatggca gggtgttttacgttatttaaTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 62) NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagct acaaagataaggcttcatgccgaaatcaacaccctgtcattttatggca gggtgtTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 63) NNNNNNNNNNNNNNNNNNNNgttattgtactctcaagatttaGAAAtaa atcttcagaagctacaatgataaggcttcatgccgaaatcaacaccctg tcattttatggcagggtgttttcgttatttaaTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Example chimeric RNA for S. thermophilus LMD-9

CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 64) NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagct acaatgataaggcttcatgccgaaatcaacaccctgtcattttatggca gggtgttttcgttatttaaTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM of NNAGAAW) (SEQ ID NO: 65) NNNNNNNNNNNNNNNNNNNNgttattgactctcaGAAAtgcagaagcta caatgataaggcttcatgccgaaatcaacaccctgtcattttatggcag ggtgTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Example chimeric RNA for S. thermophilus LMD-9 CRISPR3 Cas9 (with PAM of NGGNG) (SEQ ID NO: 66) NNNNNNNNNNNNNNNNNNNNgttttagagctgtgGAAAcacagcgagtt aaaataaggcttagtccgtactcaacttgaaaaggtggcaccgattcgg tgtTTTTTT (N = guide sequence; first underline = tracr mate sequence; second underline = tracr sequence; bold = terminator) Codon-optimized version of Cas9 from S. thermophilus LMD-9 CRISPR3 locus (with an NLS at both 5' and 3' ends) (SEQ ID NO: 67) ATGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAA AGACCAAGCCCTACAGCATCGGCCTGGACATCGGCACCAATAGCGTGGG CTGGGCCGTGACCACCGACAACTACAAGGTGCCCAGCAAGAAAATGAAG GTGCTGGGCAACACCTCCAAGAAGTACATCAAGAAAAACCTGCTGGGCG TGCTGCTGTTCGACAGCGGCATTACAGCCGAGGGCAGACGGCTGAAGAG AACCGCCAGACGGCGGTACACCCGGCGGAGAAACAGAATCCTGTATCTG CAAGAGATCTTCAGCACCGAGATGGCTACCCTGGACGACGCCTTCTTCC AGCGGCTGGACGACAGCITCCTGGTGCCCGACGACAAGCGGGACAGCAA GTACCCCATCTTCGGCAACCTGGTGGAAGAGAAGGCCTACCACGACGAG TTCCCCACCATCTACCACCTGAGAAAGTACCTGGCCGACAGCACCAAGA AGGCCGACCTGAGACTGGTGTATCTGGCCCTGGCCCACATGATCAAGTA CCGGGGCCACTTCCTGATCGAGGGCGAGTTCAACAGCAAGAACAACGAC ATCCAGAAGAACTTCCAGGACTTCCTGGACACCTACAACGCCATCTTCG AGAGCGACCTGTCCCTGGAAAACAGCAAGCAGCTGGAAGAGATCGTGAA GGACAAGATCAGCAAGCTGGAAAAGAAGGACCGCATCCTGAAGCTGTTC CCCGGCGAGAAGAACAGCGGAATCTTCAGCGAGTTTCTGAAGCTGATCG TGGGCAACCAGGCCGACTTCAGAAAGTGCTTCAACCTGGACGAGAAAGC CAGCCTGCACTTCAGCAAAGAGAGCTACGACGAGGACCTGGAAACCCTG CTGGGATATATCGGCGACGACTACAGCGACGTGTTCCTGAAGGCCAAGA AGCTGTACGACGCTATCCTGCTGAGCGGCTTCCTGACCGTGACCGACAA CGAGACAGAGGCCCCACTGAGCAGCGCCATGATTAAGCGGTACAACGAG CACAAAGAGGATCTGGCTCTGCTGAAAGAGTACATCCGGAACATCAGCC TGAAAACCTACAATGAGGTGTTCAAGGACGACACCAAGAACGGCTACGC CGGCTACATCGACGGCAAGACCAACCAGGAAGATTTCTATGTGTACCTG AAGAAGCTGCTGGCCGAGTTCGAGGGGGCCGACTACTTTCTGGAAAAAA TCGACCGCGAGGATTTCCTGCGGAAGCAGCGGACCTTCGACAACGGCAG CATCCCCTACCAGATCCATCTGCAGGAAATGCGGGCCATCCTGGACAAG CAGGCCAAGTTCTACCCATTCCTGGCCAAGAACAAAGAGCGGATCGAGA AGATCCTGACCTTCCGCATCCCTTACTACGTGGGCCCCCTGGCCAGAGG CAACAGCGATTTTGCCTGGTCCATCCGGAAGCGCAATGAGAAGATCACC CCCTGGAACTTCGAGGACGTGATCGACAAAGAGTCCAGCGCCGAGGCCT TCATCAACCGGATGACCAGCTTCGACCTGTACCTGCCCGAGGAAAAGGT GCTGCCCAAGCACAGCCTGCTGTACGAGACATTCAATGTGTATAACGAG CTGACCAAAGTGCGGTTTATCGCCGAGTCTATGCGGGACTACCAGTTCC TGGACTCCAAGCAGAAAAAGGACATCGTGCGGCTGTACTTCAAGGACAA GCGGAAAGTGACCGATAAGGACATCATCGAGTACCTGCACGCCATCTAC GGCTACGATGGCATCGAGCTGAAGGGCATCGAGAAGCAGTTCAACTCCA GCCTGAGCACATACCACGACCTGCTGAACATTATCAACGACAAAGAATT TCTGGACGACTCCAGCAACGAGGCCATCATCGAAGAGATCATCCACACC CTGACCATCTTTGAGGACCGCGAGATGATCAAGCAGCGGCTGAGCAAGT TCGAGAACATCTTCGACAAGAGCGTGCTGAAAAAGCTGAGCAGACGGCA CTACACCGGCTGGGGCAAGCTGAGCGCCAAGCTGATCAACGGCATCCGG GACGAGAAGTCCGGCAACACAATCCTGGACTACCTGATCGACGACGGCA TCAGCAACCGGAACTTCATGCAGCTGATCCACGACGACGCCCTGAGCTT CAAGAAGAAGATCCAGAAGGCCCAGATCATCGGGGACGAGGACAAGGGC AACATCAAAGAAGTCGTGAAGTCCCTGCCCGGCAGCCCCGCCATCAAGA AGGGAATCCTGCAGAGCATCAAGATCGTGGACGAGCTCGTGAAAGTGAT GGGCGGCAGAAAGCCCGAGAGCATCGTGGTGGAAATGGCTAGAGAGAAC CAGTACACCAATCAGGGCAAGAGCAACAGCCAGCAGAGACTGAAGAGAC TGGAAAAGTCCCTGAAAGAGCTGGGCAGCAAGATTCTGAAAGAGAATAT CCCTGCCAAGCTGTCCAAGATCGACAACAACGCCCTGCAGAACGACCGG CTGTACCTGTACTACCTGCAGAATGGCAAGGACATGTATACAGGCGACG ACCTGGATATCGACCGCCTGAGCAACTACGACATCGACCATATTATCCC CCAGGCCTTCCTGAAAGACAACAGCATTGACAACAAAGTGCTGGTGTCC TCCGCCAGCAACCGCGGCAAGTCCGATGATGTGCCCAGCCTGGAAGTCG TGAAAAAGAGAAAGACCTTCTGGTATCAGCTGCTGAAAAGCAAGCTGAT TAGCCAGAGGAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTG AGCCCTGAAGATAAGGCCGGCTTCATCCAGAGACAGCTGGTGGAAACCC GGCAGATCACCAAGCACGTGGCCAGACTGCTGGATGAGAAGTTTAACAA CAAGAAGGACGAGAACAACCGGGCCGTGCGGACCGTGAAGATCATCACC CTGAAGTCCACCCTGGTGTCCCAGTTCCGGAAGGACTTCGAGCTGTATA AAGTGCGCGAGATCAATGACTTTCACCACGCCCACGACGCCTACCTGAA TGCCGTGGTGGCTTCCGCCCTGCTGAAGAAGTACCCTAAGCTGGAACCC GAGTTCGTGTACGGCGACTACCCCAAGTACAACTCCTTCAGAGAGCGGA AGTCCGCCACCGAGAAGGTGTACTTCTACTCCAACATCATGAATATCTT TAAGAAGTCCATCTCCCTGGCCGATGGCAGAGTGATCGAGCGGCCCCTG ATCGAAGTGAACGAAGAGACAGGCGAGAGCGTGTGGAACAAAGAAAGCG ACCTGGCCACCGTGCGGCGGGTGCTGAGTTATCCTCAAGTGAATGTCGT GAAGAAGGTGGAAGAACAGAACCACGGCCTGGATCGGGGCAAGCCCAAG GGCCTGTTCAACGCCAACCTGTCCAGCAAGCCTAAGCCCAACTCCAACG AGAATCTCGTGGGGGCCAAAGAGTACCTGGACCCTAAGAAGTACGGCGG ATACGCCGGCATCTCCAATAGCTTCACCGTGCTCGTGAAGGGCACAATC GAGAAGGGCGCTAAGAAAAAGATCACAAACGTGCTGGAATTTCAGGGGA TCTCTATCCTGGACCGGATCAACTACCGGAAGGATAAGCTGAACTTTCT GCTGGAAAAAGGCTACAAGGACATTGAGCTGATTATCGAGCTGCCTAAG TACTCCCTGTTCGAACTGAGCGACGGCTCCAGACGGATGCTGGCCTCCA TCCTGTCCACCAACAACAAGCGGGGCGAGATCCACAAGGGAAACCAGAT CTTCCTGAGCCAGAAATTTGTGAAACTGCTGTACCACGCCAAGCGGATC TCCAACACCATCAATGAGAACCACCGGAAATACGTGGAAAACCACAAGA AAGAGTTTGAGGAACTGTTCTACTACATCCTGGAGTTCAACGAGAACTA TGTGGGAGCCAAGAAGAACGGCAAACTGCTGAACTCCGCCTTCCAGAGC TGGCAGAACCACAGCATCGACGAGCTGTGCAGCTCCTTCATCGGCCCTA CCGGCAGCGAGCGGAAGGGACTGTTTGAGCTGACCTCCAGAGGCTCTGC CGCCGACTTTGAGTTCCTGGGAGTGAAGATCCCCCGGTACAGAGACTAC ACCCCCTCTAGTCTGCTGAAGGACGCCACCCTGATCCACCAGAGCGTGA CCGGCCTGTACGAAACCCGGATCGACCTGGCTAAGCTGGGCGAGGGAAA GCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAATAA

Example 5

RNA-Guided Editing of Bacterial Genomes Using CRLSPR-Cas Systems

[0238] Applicants used the CRISPR-associated endonuclease Cas9 to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relied on Cas9-directed cleavage at the targeted site to kill unmutated cells and circumvented the need for selectable markers or counter-selection systems. Cas9 specificity was reprogrammed by changing the sequence of short CRISPR RNA (crRNA) to make single- and multi-nucleotide changes carried on editing templates. Simultaneous use of two crRNAs enabled multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that survived Cas9 cleavage contained the desired mutation, and 65% when used in combination with recombineering in E. coli. Applicants exhaustively analyzed Cas9 target requirements to define the range of targetable sequences and showed strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.

[0239] The understanding of gene function depends on the possibility of altering DNA sequences within the cell in a controlled fashion. Site-specific mutagenesis in eukaryotes is achieved by the use of sequence-specific nucleases that promote homologous recombination of a template DNA containing the mutation of interest. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and homing meganucleases can be programmed to cleave genomes in specific locations, but these approaches require engineering of new enzymes for each target sequence. In prokaryotic organisms, mutagenesis methods either introduce a selection marker in the edited locus or require a two-step process that includes a counter-selection system. More recently, phage recombination proteins have been used for recombineering, a technique that promotes homologuous recombination of linear DNA or oligonucleotides. However, because there is no selection of mutations, recombineering efficiency can be relatively low (0.1-10% for point mutations down to 10.sup.-5-10.sup.-6 for larger modifications), in many cases requiring the screening of a large number of colonies. Therefore new technologies that are affordable, easy to use and efficient are still in need for the genetic engineering of both eukaryotic and prokaryotic organisms.

[0240] Recent work on the CRISPR (clustered, regularly interspaced, short palindromic repeats) adaptive immune system of prokaryotes has led to the identification of nucleases whose sequence specificity is programmed by small RNAs. CRISPR loci are composed of a series of repeats separated by `spacer` sequences that match the genomes of bacteriophages and other mobile genetic elements. The repeat-spacer array is transcribed as a long precursor and processed within repeat sequences to generate small crRNA that specify the target sequences (also known as protospacers) cleaved by CRISPR systems. Essential for cleavage is the presence of a sequence motif immediately downstream of the target region, known as the protospacer-adjacent motif (PAM). CRISPR-associated (cas) genes usually flank the repeat-spacer array and encode the enzymatic machinery responsible for crRNA biogenesis and targeting. Cas9 is a dsDNA endonuclease that uses a crRNA guide to specify the site of cleavage. Loading of the crRNA guide onto Cas9 occurs during the processing of the crRNA precursor and requires a small RNA antisense to the precursor, the tracrRNA, and RNAse III. In contrast to genome editing with ZFNs or TALENs, changing Cas9 target specificity does not require protein engineering but only the design of the short crRNA guide.

[0241] Applicants recently showed in S. pneumoniae that the introduction of a CRISPR system targeting a chromosomal locus leads to the killing of the transformed cells. It was observed that occasional survivors contained mutations in the target region, suggesting that Cas9 dsDNA endonuclease activity against endogenous targets could be used for genome editing. Applicants showed that marker-less mutations can be introduced through the transformation of a template DNA fragment that will recombine in the genome and eliminate Cas9 target recognition. Directing the specificity of Cas9 with several different crRNAs allows for the introduction of multiple mutations at the same time. Applicants also characterized in detail the sequence requirements for Cas9 targeting and show that the approach can be combined with recombineering for genome editing in E. coli.

[0242] RESULTS: Genome editing by Cas9 cleavage of a chromosomal target

[0243] S. pneumoniae strain crR6 contains a Cas9-based CRISPR system that cleaves a target sequence present in the bacteriophage .phi.8232.5. This target was integrated into the srtA chromosomal locus of a second strain R6.sup.8232.5. An altered target sequence containing a mutation in the PAM region was integrated into the srtA locus of a third strain R6.sup.370.1, rendering this strain `immune` to CRISPR cleavage (FIG. 28a). Applicants transformed R6.sup.8232.5 and R6.sup.370.1 cells with genomic DNA from crR6 cells, expecting that successful transformation of R6.sup.8232.5 cells should lead to cleavage of the target locus and cell death. Contrary to this expectation, Applicants isolated R6.sup.8232.5 transformants, albeit with approximately 10-fold less efficiency than R6.sup.370.1 transformants (FIG. 28b). Genetic analysis of eight R6.sup.8232.5 transformants (FIG. 28) revealed that the great majority are the product of a double recombination event that eliminates the toxicity of Cas9 targeting by replacing the .phi.8232.5 target with the crR6 genome's wild-type srtA locus, which does not contain the protospacer required for Cas9 recognition. These results were proof that the concurrent introduction of a CRISPR system targeting a genomic locus (the targeting construct) together with a template for recombination into the targeted locus (the editing template) led to targeted genome editing (FIG. 23a).

[0244] To create a simplified system for genome editing, Applicants modified the CRISPR locus in strain crR6 by deleting cas1, cas2 and csn2, genes which have been shown to be dispensable for CRISPR targeting, yielding strain crR6M (FIG. 28a). This strain retained the same properties of crR6 (FIG. 28b). To increase the efficiency of Cas9-based editing and demonstrate that a template DNA of choice can be used to control the mutation introduced, Applicants co-transformed R6.sup.8232.5 cells with PCR products of the wild-type srIA gene or the mutant R6.sup.370.1 target, either of which should be resistant to cleavage by Cas9. This resulted in a 5- to 10-fold increase of the frequency of transformation compared with genomic crR6 DNA alone (FIG. 23b). The efficiency of editing was also substantially increased, with 8/8 transformants tested containing a wild-type srtA copy and 7/8 containing the PAM mutation present in the R6.sup.370.1 target (FIG. 23b and FIG. 29a). Taken together, these results showed the potential of genome editing assisted by Cas9.

[0245] Analysis of Cas9 Target Requirements:

[0246] To introduce specific changes in the genome, one must use an editing template carrying mutations that abolish Cas9-mediated cleavage, thereby preventing cell death. This is easy to achieve when the deletion of the target or its replacement by another sequence (gene insertion) is sought. When the goal is to produce gene fusions or to generate single-nucleotide mutations, the abolishment of Cas9 nuclease activity will only be possible by introducing mutations in the editing template that alter either the PAM or the protospacer sequences. To determine the constraints of CRISPR-mediated editing, Applicants performed an exhaustive analysis of PAM and protospacer mutations that abrogate CRISPR targeting.

[0247] Previous studies proposed that S. pyogenes Cas9 requires an NGG PAM immediately downstream of the protospacer. However, because only a very limited number of PAM-inactivating mutations have been described so far, Applicants conducted a systematic analysis to find all 5-nucleotide sequences following the protospacer that eliminate CRISPR cleavage. Applicants used randomized oligonucleotides to generate all possible 1,024 PAM sequences in a heterogeneous PCR product that was transformed into crR6 or R6 cells. Constructs carrying functional PAMs were expected to be recognized and destroyed by Cas9 in crR6 but not R6 cells (FIG. 24a). More than 2.times.10' colonies were pooled together to extract DNA for use as template for the co-amplification of all targets. PCR products were deep sequenced and found to contain all 1,024 sequences, with coverage ranging from 5 to 42,472 reads (See section "Analysis of deep sequencing data"). The functionality of each PAM was estimated by the relative proportion of its reads in the crR6 sample over the R6 sample. Analysis of the first three bases of the PAM, averaging over the two last bases, clearly showed that the NGG pattern was under-represented in crR6 transformants (FIG. 24b). Furthermore, the next two bases had no detectable effect on the NGG PAM (See section "Analysis of deep sequencing data"), demonstrating that the NGGNN sequence was sufficient to license Cas9 activity. Partial targeting was observed for NAG PAM sequences (FIG. 24b). Also the NNGGN pattern partially inactivated CRISPR targeting (Table G), indicating that the NGG motif can still be recognized by Cas9 with reduced efficiency when shifted by 1 bp. These data shed light onto the molecular mechanism of Cas9 target recognition, and they revealed that NGG (or CCN on the complementary strand) sequences are sufficient for Cas9 targeting and that NGG to NAG or NNGGN mutations in the editing template should be avoided. Owing to the high frequency of these tri-nucleotide sequences (once every 8 bp), this means that almost any position of the genome can be edited. Indeed, Applicants tested ten randomly chosen targets carrying various PAMs and all were found to be functional (FIG. 30).

[0248] Another way to disrupt Cas9-mediated cleavage is to introduce mutations in the protospacer region of the editing template. It is known that point mutations within the `seed sequence` (the 8 to 10 protospacer nucleotides immediately adjacent to the PAM) can abolish cleavage by CRISPR nucleases. However, the exact length of this region is not known, and it is unclear whether mutations to any nucleotide in the seed can disrupt Cas9 target recognition. Applicants followed the same deep sequencing approach described above to randomize the entire protospacer sequence involved in base pair contacts with the crRNA and to determine all sequences that disrupt targeting. Each position of the 20 matching nucleotides (14) in the spc1 target present in R6.sup.8232.5 cells (FIG. 23a) was randomized and transformed into crR6 and R6 cells (FIG. 24a). Consistent with the presence of a seed sequence, only mutations in the 12 nucleotides immediately upstream of the PAM abrogated cleavage by Cas9 (FIG. 24c). However, different mutations displayed markedly different effects. The distal (from the PAM) positions of the seed (12 to 7) tolerated most mutations and only one particular base substitution abrogated targeting. In contrast, mutations to any nucleotide in the proximal positions (6 to 1, except 3) eliminated Cas9 activity, although at different levels for each particular substitution. At position 3, only two substitutions affected CRISPR activity and with different strength. Applicants concluded that, although seed sequence mutations can prevent CRISPR targeting, there are restrictions regarding the nucleotide changes that can be made in each position of the seed. Moreover, these restrictions can most likely vary for different spacer sequences. Therefore Applicants believe that mutations in the PAM sequence, if possible, should be the preferred editing strategy. Alternatively, multiple mutations in the seed sequence may be introduced to prevent Cas9 nuclease activity.

[0249] Cas9-Mediated Genome Editing in S. pneumonia:

[0250] To develop a rapid and efficient method for targeted genome editing, Applicants engineered strain crR6Rk, a strain in which spacers can be easily introduced by PCR (FIG. 33). Applicants decided to edit the .beta.-galactosidase (bgaA) gene of S. pneumoniae, whose activity can be easily measured. Applicants introduced alanine substitutions of amino acids in the active site of this enzyme: R481A (R-.A) and N563A,E564A (NE.fwdarw.AA) mutations. To illustrate different editing strategies, Applicants designed mutations of both the PAM sequence and the protospacer seed. In both cases the same targeting construct with a crRNA complementary to a region of the .beta.-galactosidase gene that is adjacent to a TGG PAM sequence (CCA in the complementary strand, FIG. 26) was used. The R.fwdarw.A editing template created a three-nucleotide mismatch on the protospacer seed sequence (CGT to GCA, also introducing a BtgZI restriction site). In the NE.fwdarw.AA editing template Applicants simultaneously introduced a synonymous mutation that created an inactive PAM (TGG to TTG) along with mutations that are 218 nt downstream of the protospacer region (AAT GAA to GCT GCA, also generating a TseI restriction site). This last editing strategy demonstrated the possibility of using a remote PAM to make mutations in places where a proper target may be hard to choose. For example, although the S. pneumoniae R6 genome, which has a 39.7% GC content, contains on average one PAM motif every 12 bp, some PAM motifs are separated by up to 194 bp (FIG. 33). In addition Applicants designed a .DELTA.bgaA in-frame deletion of 6,664 bp. In all three cases, co-transformation of the targeting and editing templates produced 10-times more kanamycin-resistant cells than co-transformation with a control editing template containing wild-type bgaA sequences (FIG. 25b). Applicants genotyped 24 transformants (8 for each editing experiment) and found that all but one incorporated the desired change (FIG. 25c). DNA sequencing also confirmed not only the presence of the introduced mutations but also the absence of secondary mutations in the target region (FIG. 29b,c). Finally, Applicants measured .beta.-galactosidase activity to confirm that all edited cells displayed the expected phenotype (FIG. 25d).

[0251] Cas9-mediated editing can also be used to generate multiple mutations for the study of biological pathways. Applicants decided to illustrate this for the sortase-dependent pathway that anchors surface proteins to the envelope of Gram-positive bacteria. Applicants introduced a sortase deletion by co-transformation of a chloramphenicol-resistant targeting construct and a .DELTA.srtA editing template (FIG. 33a,b), followed by a .DELTA.bgaA deletion using a kanamycin-resistant targeting construct that replaced the previous one. In S. pneumoniae, .beta.-galactosidase is covalently linked to the cell wall by sortase. Therefore, deletion of srtA results in the release of the surface protein into the supernatant, whereas the double deletion has no detectable .beta.-galactosidase activity (FIG. 34c). Such a sequential selection can be iterated as many times as required to generate multiple mutations.

[0252] These two mutations may also be introduced at the same time. Applicants designed a targeting construct containing two spacers, one matching srtA and the other matching bgaA, and co-transformed it with both editing templates at the same time (FIG. 25e). Genetic analysis of transformants showed that editing occurred in 6/8 cases (FIG. 25f). Notably, the remaining two clones each contained either a .DELTA.srtA or a .DELTA.bgaA deletion, suggesting the possibility of performing combinatorial mutagenesis using Cas9. Finally, to eliminate the CRISPR sequences, Applicants introduced a plasmid containing the bgaA target and a spectinomycin resistance gene along with genomic DNA from the wild-type strain R6. Spectinomycin-resistant transformants that retain the plasmid eliminated the CRISPR sequences (FIG. 34a,d).

[0253] Mechanism and Efficiency of Editing:

[0254] To understand the mechanisms underlying genome editing with Cas9, Applicants designed an experiment in which the editing efficiency was measured independently of Cas9 cleavage. Applicants integrated the ermAM erythromycin resistance gene in the srtA locus, and introduced a premature stop codon using Cas9-mediated editing (FIG. 33). The resulting strain (JEN53) contains an ermAM(stop) allele and is sensitive to erythromycin. This strain may be used to assess the efficiency at which the ermAM gene is repaired by measuring the fraction of cells that restore antibiotic resistance with or without the use of Cas9 cleavage. JEN53 was transformed with an editing template that restores the wild-type allele, together with either a kanamycin-resistant CRISPR construct targeting the ermAM(stop) allele (CRISPR::ermAM(stop)) or a control construct without a spacer (CRISPR::O) (FIG. 26a,b). In the absence of kanamycin selection, the fraction of edited colonies was on the order of 10.sup.-2 (erythromycin-resistant cfu/total cfu) (FIG. 26c), representing the baseline frequency of recombination without Cas9-mediated selection against unedited cells. However, if kanamycin selection was applied and the control CRISPR construct was co-transformed, the fraction of edited colonies increased to about 10.sup.-1 (kanamycin- and erythromycin-resistant cfu/kanamycin-resistant cfu) (FIG. 26c). This result shows that selection for the recombination of the CRISPR locus co-selected for recombination in the ermAM locus independently of Cas9 cleavage of the genome, suggesting that a subpopulation of cells is more prone to transformation and/or recombination. Transformation of the CRISPR::ermAM(stop) construct followed by kanamycin selection resulted in an increase of the fraction of erythromycin-resistant, edited cells to 99% (FIG. 26c). To determine if this increase is caused by the killing of non-edited cells, Applicants compared the kanamycin-resistant colony forming units (cfu) obtained after co-transformation of JEN53 cells with the CRISPR::ermAM(stop) or CRISPR::O constructs.

[0255] Applicants counted 5.3 times less kanamycin-resistant colonies after transformation of the ermAM(stop) construct (2.5.times.10.sup.4/4.7.times.10.sup.3, FIG. 35a), a result that suggests that indeed targeting of a chromosomal locus by Cas9 leads to the killing of non-edited cells. Finally, because the introduction of dsDNA breaks in the bacterial chromosome is known to trigger repair mechanisms that increase the rate of recombination of the damaged DNA, Applicants investigated whether cleavage by Cas9 induces recombination of the editing template. Applicants counted 2.2 times more colonies after co-transformation with the CRISPR::erm(stop) construct than with the CRISPR::O construct (FIG. 26d), indicating that there was a modest induction of recombination. Taken together, these results showed that co-selection of transformable cells, induction of recombination by Cas9-mediated cleavage and selection against non-edited cells, each contributed to the high efficiency of genome editing in S. pneumoniae.

[0256] As cleavage of the genome by Cas9 should kill non-edited cells, one would not expect to recover any cells that received the kanamycin resistance-containing Cas9 cassette but not the editing template. However, in the absence of the editing template Applicants recovered many kanamycin-resistant colonies after transformation of the CRISPR::ermAM(stop) construct (FIG. 35a). These cells that `escape` CRISPR-induced death produced a background that determined a limit of the method. This background frequency may be calculated as the ratio of CRISPR::ermAM(stop)/CRISPR::O cfu, 2.6.times.10.sup.-1 (7.1.times.10.sup.1/2.7.times.10.sup.4) in this experiment, meaning that if the recombination frequency of the editing template is less than this value, CRISPR selection may not efficiently recover the desired mutants above the background. To understand the origin of these cells, Applicants genotyped 8 background colonies and found that 7 contained deletions of the targeting spacer (FIG. 35b) and one harbored a presumably inactivating mutation in Cas9 (FIG. 35c).

[0257] Genome Editing with Cas9 in E. coli:

[0258] The activation of Cas9 targeting through the chromosomal integration of a CRISPR-Cas system is only possible in organisms that are highly recombinogenic. To develop a more general method that is applicable to other microbes, Applicants decided to perform genome editing in E. coli using a plasmid-based CRISPR-Cas system. Two plasmids were constructed: a pCas9 plasmid carrying the tracrRNA, Cas9 and a chloramphenicol resistance cassette (FIG. 36), and a pCRISPR kanamycin-resistant plasmid carrying the array of CRISPR spacers. To measure the efficiency of editing independently of CRISPR selection, Applicants sought to introduce an A to C transversion in the rpsL gene that confers streptomycin resistance. Applicants constructed a pCRISPR::rpsL plasmid harboring a spacer that would guide Cas9 cleavage of the wild-type, but not the mutant rpsL allele (FIG. 27b). The pCas9 plasmid was first introduced into E. coli MG1655 and the resulting strain was co-transformed with the pCRISPR::rpsL plasmid and W542, an editing oligonucleotide containing the A to C mutation. streptomycin-resistant colonies after transformation of the pCRISPR::rpsL plasmid were only recovered, suggesting that Cas9 cleavage induces recombination of the oligonucleotide (FIG. 37). However, the number of streptomycin-resistant colonies was two orders of magnitude lower than the number of kanamycin-resistant colonies, which are presumably cells that escape cleavage by Cas9. Therefore, in these conditions, cleavage by Cas9 facilitated the introduction of the mutation, but with an efficiency that was not enough to select the mutant cells above the background of `escapers`.

[0259] To improve the efficiency of genome editing in E. coli, Applicants applied their CRISPR system with recombineering, using Cas9-induced cell death to select for the desired mutations. The pCas9 plasmid was introduced into the recombineering strain HME63 (31), which contains the Gam, Exo and Beta functions of the .quadrature.-red phage. The resulting strain was co-transformed with the pCRISPR::rpsL plasmid (or a pCRISPR::O control) and the W542 oligonucleotide (FIG. 27a). The recombineering efficiency was 5.3.times.10.sup.-5, calculated as the fraction of total cells that become streptomycin-resistant when the control plasmid was used (FIG. 27c). In contrast, transformation with the pCRISPR::rpsL plasmid increased the percentage of mutant cells to 65.+-.14% (FIGS. 27c and 29f). Applicants observed that the number of cfu was reduced by about three orders of magnitude after transformation of the pCRISPR::rpsL plasmid than the control plasmid (4.8.times.10.sup.5/5.3.times.10.sup.2, FIG. 38a), suggesting that selection results from CRISPR-induced death of non-edited cells. To measure the rate at which Cas9 cleavage was inactivated, an important parameter of Applicants' method, Applicants transformed cells with either pCRISPR::rpsL or the control plasmid without the W542 editing oligonucleotide (FIG. 38a). This background of CRISPR `escapers`, measured as the ratio of pCRISPR::rpsL/pCRISPR::O cfu, was 2.5.times.10.sup.-4 (1.2.times.10.sup.2/4.8.times.10.sup.5). Genotyping eight of these escapers revealed that in all cases there was a deletion of the targeting spacer (FIG. 38b). This background was higher than the recombineering efficiency of the rpsL mutation, 5.3.times.10'5, which suggested that to obtain 65% of edited cells, Cas9 cleavage must induce oligonucleotide recombination. To confirm this, Applicants compared the number of kanamycin- and streptomycin-resistant cfu after transformation of pCRISPR::rpsL or pCRISPR::O (FIG. 27d). As in the case for S. pneumoniae, Applicants observed a modest induction of recombination, about 6.7 fold (2.0.times.10.sup.-4/3.0.times.10.sup.-5). Taken together, these results indicated that the CRISPR system provided a method for selecting mutations introduced by recombineering.

[0260] Applicants showed that CRISPR-Cas systems may be used for targeted genome editing in bacteria by the co-introduction of a targeting construct that killed wild-type cells and an editing template that both eliminated CRISPR cleavage and introduced the desired mutations. Different types of mutations (insertions, deletions or scar-less single-nucleotide substitutions) may be generated. Multiple mutations may be introduced at the same time. The specificity and versatility of editing using the CRISPR system relied on several unique properties of the Cas9 endonuclease: (i) its target specificity may be programmed with a small RNA, without the need for enzyme engineering, (ii) target specificity was very high, determined by a 20 bp RNA-DNA interaction with low probability of non-target recognition, (iii) almost any sequence may be targeted, the only requirement being the presence of an adjacent NGG sequence, (iv) almost any mutation in the NGG sequence, as well as mutations in the seed sequence of the protospacer, eliminates targeting.

[0261] Applicants showed that genome engineering using the CRISPR system worked not only in highly recombinogenic bacteria such as S. pneumoniae, but also in E. coli. Results in E. coli suggested that the method may be applicable to other microorganisms for which plasmids may be introduced. In E. coli, the approach complements recombineering of mutagenic oligonucleotides. To use this methodology in microbes where recombineering is not a possible, the host homologous recombination machinery may be used by providing the editing template on a plasmid. In addition, because accumulated evidence indicates that CRISPR-mediated cleavage of the chromosome leads to cell death in many bacteria and archaea, it is possible to envision the use of endogenous CRISPR-Cas systems for editing purposes.

[0262] In both S. pneumoniae and E. coli, Applicants observed that although editing was facilitated by a co-selection of transformable cells and a small induction of recombination at the target site by Cas9 cleavage, the mechanism that contributed the most to editing was the selection against non-edited cells. Therefore the major limitation of the method was the presence of a background of cells that escape CRISPR-induced cell death and lack the desired mutation. Applicants showed that these `escapers` arose primarily through the deletion of the targeting spacer, presumably after the recombination of the repeat sequences that flank the targeting spacer. Future improvements may focus on the engineering of flanking sequences that can still support the biogenesis of functional crRNAs but that are sufficiently different from one another to eliminate recombination. Alternatively, the direct transformation of chimeric crRNAs may be explored. In the particular case of E. coli, the construction of the CRISPR-Cas system was not possible if this organism was also used as a cloning host. Applicants solved this issue by placing Cas9 and the tracrRNA on a different plasmid than the CRISPR array. The engineering of an inducible system may also circumvent this limitation.

[0263] Although new DNA synthesis technologies provide the ability to cost-effectively create any sequence with a high throughput, it remains a challenge to integrate synthetic DNA in living cells to create functional genomes. Recently, the co-selection MAGE strategy was shown to improve the mutation efficiency of recombineering by selecting a subpopulation of cells that has an increased probability to achieve recombination at or around a given locus. In this method, the introduction of selectable mutations is used to increase the chances of generating nearby non-selectable mutations. As opposed to the indirect selection provided by this strategy, the use of the CRISPR system makes it possible to directly select for the desired mutation and to recover it with a high efficiency. These technologies add to the toolbox of genetic engineers, and together with DNA synthesis, they may substantially advance both the ability to decipher gene function and to manipulate organisms for biotechnological purposes. Two other studies also relate to CRISPR-assisted engineering of mammalian genomes. It is expected that these crRNA-directed genome editing technologies may be broadly useful in the basic and medical sciences.

[0264] Strains and Culture Conditions.

[0265] S. pneumoniae strain R6 was provided by Dr. Alexander Tomasz. Strain crR6 was generated in a previous study. Liquid cultures of S. pneumoniae were grown in THYE medium (30 g/l Todd-Hewitt agar, 5 g/l yeast extract). Cells were plated on tryptic soy agar (TSA) supplemented with 5% defibrinated sheep blood. When appropriate, antibiotics were added as followings: kanamycin (400 .mu.g/ml), chloramphenicol (5 .mu.g/ml), erythromycin (1 .mu.g/ml) streptomycin (100 .mu.g/ml) or spectinomycin (100 .mu.g/ml). Measurements of .beta.-galactosidase activity were made using the Miller assay as previously described.

[0266] E. coli strains MG1655 and HME63 (derived from MG1655, .DELTA.(argF-lac) U169.lamda. c1857 .DELTA.cro-bioA galK tyr 145 UAG mutS< >amp) (31) were provided by Jeff Roberts and Donald Court, respectively. Liquid cultures of E. coli were grown in LB medium (Difco). When appropriate, antibiotics were added as followings: chloramphenicol (25 .mu.g/ml), kanamycin (25 .mu.g/ml) and streptomycin (50 .mu.g/ml).

[0267] S. pneunmoniae Transformation.

[0268] Competent cells were prepared as described previously (23). For all genome editing transformations, cells were gently thawed on ice and resuspended in 10 volumes of M2 medium supplemented with 100 ng/ml of competence-stimulating peptide CSP1(40), and followed by addition of editing constructs (editing constructs were added to cells at a final concentration between 0.7 ng/.mu.l to 2.5 .mu.g/ul). Cells were incubated 20 min at 37.degree. C. before the addition of 2 .mu.l of targeting constructs and then incubated 40 min at 37 .mu.C. Serial dilutions of cells were plated on the appropriate medium to determine the colony forming units (cfu) count.

[0269] E. coli Lambda-Red Recombineering.

[0270] Strain HME63 was used for all recombineering experiments. Recombineering cells were prepared and handled according to a previously published protocol (6). Briefly, a 2 ml overnight culture (LB medium) inoculated from a single colony obtained from a plate was grown at 30.degree. C. The overnight culture was diluted 100-fold and grown at 30.degree. C. with shaking (200 rpm) until the OD.sub.600 is from 0.4-0.5 (approximately 3 hrs). For Lambda-red induction, the culture was transferred to a 42.degree. C. water bath to shake at 200 rpm for 15 min. Immediately after induction, the culture was swirled in an ice-water slurry and chilled on ice for 5-10 min. Cells were then washed and aliquoted according to the protocol. For electro-transformation, 50 .mu.l of cells were mixed with 1mM of salt-free oligos (IDT) or 100-150 ng of plasmid DNA (prepared by QIAprep Spin Miniprep Kit, Qiagen). Cells were electroporated using 1 mm Gene Pulser cuvette (Bio-rad) at 1.8 kV and were immediately resuspended in 1 ml of room temperature LB medium. Cells were recovered at 30.degree. C. for 1-2 hrs before being plated on LB agar with appropriate antibiotic resistance and incubated at 32.degree. C. overnight.

[0271] Preparation of S. pneumoniae Genomic DNA.

[0272] For transformation purposes, S. pneumoniae genomic DNA was extracted using the Wizard Genomic DNA Purification Kit, following instructions provided by the manufacturer (Promega). For genotyping purposes, 700 ul of overnight S. pneumoniae cultures were pelleted, resuspended in 60 ul of lysozyme solution (2 mg/ml) and incubated 30 min at 37.degree. C. The genomic DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen).

[0273] Strain Construction.

[0274] All primers used in this study are provided in Table G. To generate S. pneumoniae crR6M, an intermediate strain, LAM226, was made. In this strain the alphA-3 gene (providing kanamycin resistance) adjacent to the CRISPR array of S. pneumoniae crR6 strain was replaced by a cat gene (providing chloramphenicol resistance). Briefly, crR6 genomic DNA was amplified using primers L448/L444 and L447/L481, respectively. The cat gene was amplified from plasmid pC194 using primers L445/L446. Each PCR product was gel-purified and all three were fused by SOEing PCR with primers L448/L481. The resulting PCR product was transformed into competent S. pneumoniae crR6 cells and chloramphenicol-resistant transformants were selected. To generate S. pneumoniae crR6M, S. pneumoniae crR6 genomic DNA was amplified by PCR using primers L409/L488 and L448/L481, respectively. Each PCR product was gel-purified and they were fused by SOEing PCR with primers L409/L481. The resulting PCR product was transformed into competent S. pneumoniae LAM226 cells and kanamycin-resistant transformants were selected.

[0275] To generate S. pneumoniae crR6Rc, S. pneumoniae crR6M genomic DNA was amplified by PCR using primers L430/W286, and S. pneumoniae LAM226 genomic DNA was amplified by PCR using primers W288/L481. Each PCR product was gel-purified and they were fused by SOEing PCR with primers L430/L481. The resulting PCR product was transformed into competent S. pneumoniae crR6M cells and chloramphenicol-resistant transformants were selected.

[0276] To generate S. pneumoniae crR6Rk, S. pneumoniae crR6M genomic DNA was amplified by PCR using primers L430/W286 and W287/L481, respectively. Each PCR product was gel-purified and they were fused by SOEing PCR with primers L430/L481. The resulting PCR product was transformed into competent S. pneumoniae crR6Rc cells and kanamycin-resistant transformants were selected.

[0277] To generate JEN37, S. pneumoniae crR6Rk genomic DNA was amplified by PCR using primers L430/W356 and W357/L481, respectively. Each PCR product was gel-purified and they were fused by SOEing PCR with primers L430/L481. The resulting PCR product was transformed into competent S. pneumoniae crR6Rc cells and kanamycin-resistant transformants were selected.

[0278] To generate JEN38, R6 genomic DNA was amplified using primers L422/L461 and L459/L426, respectively. The ermAM gene (specifying erythromycin resistance) was amplified from plasmid pFW15.sup.43 using primers L457/L458. Each PCR product was gel-purified and all three were fused by SOEing PCR with primers L422/L426. The resulting PCR product was transformed into competent S. pneumoniae crR6Rc cells and erythromycin-resistant transformants were selected.

[0279] S. pneumoniae JEN53 was generated in two steps. First JEN43 was constructed as illustrated in FIG. 33. JEN53 was generated by transforming genomic DNA of JEN25 into competent JEN43 cells and selecting on both chloramphenicol and erythromycin.

[0280] To generate S. pneumoniae JEN62, S. pneumoniae crR6Rk genomic DNA was amplified by PCR using primers W256/W365 and W366/L403, respectively. Each PCR product was purified and ligated by Gibson assembly. The assembly product was transformed into competent S. pneumoniae crR6Rc cells and kanamycin-resistant transformants were selected.

[0281] Plasmid Construction.

[0282] pDB97 was constructed through phosphorylation and annealing of oligonucleotides B296/B297, followed by ligation in pLZ12spec digested by EcoRI/BamHI. Applicants fully sequenced pLZ12spec and deposited its sequence in genebank (accession: KC 112384).

[0283] pDB98 was obtained after cloning the CRISPR leader sequence was cloned together with a repeat-spacer-repeat unit into pLZ12spec. This was achieved through amplification of crR6Rc DNA with primers B298/B320 and B299/B321, followed by SOEing PCR of both products and cloning in pLZ12spec with restriction sites BamHI/EcoRI. In this way the spacer sequence in pDB98 was engineered to contain two BsaI restriction sites in opposite directions that allow for the scar-less cloning of new spacers.

[0284] pDB99 to pDB108 were constructed by annealing of oligonucleotides B300/B301 (pDB99), B302/B303 (pDB100), B304/B305 (pDB01), B306/B307 (pDB102), B308/B309 (pDB103), B310/B311 (pDB104), B312/B313 (pDB105), B314/B315 (pDB106), B315/B317 (pDB107), B318/B319 (pDB108), followed by ligation in pDB98 cut by BsaI.

[0285] The pCas9 plasmid was constructed as follow. Essential CRISPR elements were amplified from Streptococcos pyogenes SF370 genomic DNA with flanking homology arms for Gibson Assembly. The tracrRNA and Cas9 were amplified with oligos HC008 and HCO10. The leader and CRISPR sequences were amplified HC011/HC014 and HC015/HC009, so that two BsaI type IIS sites were introduced in between two direct repeats to facilitate easy insertion of spacers.

[0286] pCRISPR was constructed by subcloning the pCas9 CRISPR array in pZE21-MCSI through amplification with oligos B298+B299 and restriction with EcoRI and BamHI. The rpsL targeting spacer was cloned by annealing of oligos B352+B353 and cloning in the BsaI cut pCRISPR giving pCRISPR::rpsL.

[0287] Generation of Targeting and Editing Constructs.

[0288] Targeting constructs used for genome editing were made by Gibson assembly of Left PCRs and Right PCRs (Table G). Editing constructs were made by SOEing PCR fusing PCR products A (PCR A), PCR products B (PCR B) and PCR products C (PCR C) when applicable (Table G). The CRISPR::O and CRISPR::ermAM(stop) targeting constructs were generated by PCR amplification of JEN62 and crR6 genomic DNA respectively, with oligos L409 and L481.

[0289] Generation of Targets with Randomized PAM or Protospacer Sequences.

[0290] The 5 nucleotides following the spacer 1 target were randomized through amplification of R6.sup.8232.5 genomic DNA with primers W377/L426. This PCR product was then assembled with the cat gene and the srtA upstream region that were amplified from the same template with primers L422/W376. 80 ng of the assembled DNA was used to transform strains R6 and crR6. Samples for the randomized targets were prepared using the following primers: B280-B290/L426 to randomize bases 1-10 of the target and B269-B278/L426 to randomize bases 10-20. Primers L422/B268 and L422/B279 were used to amplify the cat gene and srtA upstream region to be assembled with the first and last 10 PCR products respectively. The assembled constructs were pooled together and 30 ng was transformed in R6 and crR6. After transformation, cells were plated on chloramphenicol selection. For each sample more than 2.times.10.sup.5 cells were pooled together in 1 ml of THYE and genomic DNA was extracted with the Promega Wizard kit. Primers B250/B251 were used to amplify the target region. PCR products were tagged and run on one Illumina MiSeq paired-end lane using 300 cycles.

[0291] Analysis of Deep Sequencing Data.

[0292] Randomized PAM: For the randomized PAM experiment 3,429,406 reads were obtained for crR6 and 3,253,998 for R6. It is expected that only half of them will correspond to the PAM-target while the other half will sequence the other end of the PCR product. 1,623,008 of the crR6 reads and 1,537,131 of the R6 reads carried an error-free target sequence. The occurrence of each possible PAM among these reads is shown in supplementary file. To estimate the functionality of a PAM, its relative proportion in the crR6 sample over the R6 sample was computed and is denoted r.sub.ijklm where are one of the 4 possible bases. The following statistical model was constructed:

log(r.sub.ijklm)=.mu.b2.sub.i+b3.sub.j+b4.sub.k+b2b3.sub.i,j+b3b4.sub.j,- k+.epsilon..sub.ijklm,

[0293] where .epsilon. is the residual error, b2 is the effect of the 2.sup.nd base of the PAM, b3 of the third, b4 of the fourth, b2b3 is the interaction between the second and third bases, b3b4 between the third and fourth bases. An analysis of variance was performed:

TABLE-US-00007 Anova table Df Sum Sq Mean Sq F value Pr (>F) b3 3 151.693 50.564 601.8450 <2.2e-16 *** b2 3 90.521 30.174 359.1454 <2.2e-16 *** b4 3 1.881 0.627 7.4623 6.070e-05 *** b3:b2 9 228.940 25.438 302.7738 <2.2e-16 *** b3:b4 9 3.010 0.334 3.9809 5.227e-05 *** Residuals 996 83.680 0.084

[0294] When added to this model, b1 or b5 do not appear to be significant and other interactions than the ones included can also be discarded. The model choice was made through successive comparisons of more or less complete models using the anova method in R. Tukey's honest significance test was used to determine if pairwise differences between effects are significant.

[0295] NGGNN patterns are significantly different from all other patterns and carry the strongest effect (see table below)

[0296] In order to show that positions 1, 4 or 5 do not affect the NGGNN pattern Applicants looked at theses sequences only. Their effect appears to be normally distributed (see QQ plot in FIG. 71), and model comparisons using the anova method in R shows that the null model is the best one, i.e. there is no significant role of b1, b4 and b5.

TABLE-US-00008 Model comparison using the anova method in R for the NGGNN sequences Model 1: ratio.log~1 Model 2: ratio.log~b1 / b4 + b5 Res. Df RSS Df Sum of Sq F Pr (>F) 1 63 14.579 2 54 11.295 9 3.2336 1.7443 0.1013

[0297] Partial interference of NAGNN and NNGGN patterns

[0298] NAGNN patterns are significantly different from all other patterns but carry a much smaller effect than NGGNN (see Tukey's honest significance test below).

[0299] Finally, NTGGN and NCGGN patterns are similar and show significantly more CRISPR interference than NTGHN and NCGHN patterns (where H is A, T or C), as shown by a bonferroni adjusted pairwi se student-test.

TABLE-US-00009 Pairwise comparisons of the effect of b4 on NYGNN sequences using t tests with pooled SD Data: b4 A C G C 1.00 -- -- G 9.2e-05 2.4e-06 -- T 0.31 1.00 1.2e-08

[0300] Taken together, these results allow concluding that NNGGN patterns in general produce either a complete interference in the case of NGGGN, or a partial interference in the case of NAGGN, NTGGN or NCGGN.

[0301] Tukey multiple comparisons of means: 95% family-wise confidence level

TABLE-US-00010 diff lwr upr p adj $b2:b3 G:G-A:A -2.76475 -2.94075 -2.58875 <1E-07 G:G-C:A -2.79911 -2.97511 -2.62311 <1E-07 G:G-T:A -2.7809 -2.9569 -2.6049 <1E-07 G:G-A:C -2.81643 -2.99244 -2.64043 <1E-07 G:G-C:C -2.77903 -2.95504 -2.60303 <1E-07 G:G-G:C -2.64867 -2.82468 -2.47267 <1E-07 G:G-T:C -2.79718 -2.97319 -2.62118 <1E-07 G:G-A:G -2.67068 -2.84668 -2.49468 <1E-07 G:G-C:G -2.73525 -2.91125 -2.55925 <1E-07 G:G-T:G -2.7976 -2.62159 -2.9736 <1E-07 G:G-A:T -2.76727 -2.59127 -2.94328 <1E-07 G:G-C:T -2.84114 -2.66513 -3.01714 <1E-07 G:G-G:T -2.76409 -2.58809 -2.94009 <1E-07 G:G-T:T -2.76781 -2.59181 -2.94381 <1E-07 G:G-G:A -2.13964 -2.31565 -1.96364 <1E-07 G:A-A:A -0.62511 -0.80111 -0.4491 <1E-07 G:A-C:A -0.65947 -0.83547 -0.48346 <1E-07 G:A-T:A -0.64126 -0.46525 -0.81726 <1E-07 G:A-A:C -0.67679 -0.50078 -0.85279 <1E-07 G:A-C:C -0.63939 -0.46339 -0.81539 <1E-07 G:A-G:C -0.50903 -0.33303 -0.68503 <1E-07 G:A-T:C -0.65754 -0.48154 -0.83354 <1E-07 G:A-A:G -0.53104 -0.35503 -0.70704 <1E-07 G:A-C:G -0.59561 -0.4196 -0.77161 <1E-07 G:A-T:G -0.65795 -0.48195 -0.83396 <1E-07 G:A-A:T -0.62763 -0.45163 -0.80363 <1E-07 G:A-C:T -0.70149 -0.52549 -0.8775 <1E-07 G:A-G:T -0.62445 -0.44844 -0.80045 <1E-07 G:A-T:T -0.62817 -0.45216 -0.80417 <1E-07 $b3:b4 G:G-G:A -0.33532 -0.51133 -0.15932 <1E-07 G:G-G:C -0.18118 -0.35719 -0.00518 0.036087 G:G-G:T -0.31626 -0.14026 -0.49226 <1E-07

[0302] Randomized Target

[0303] For the randomized target experiment 540,726 reads were obtained for crR6 and 753,570 for R6. As before, only half of the reads are expected to sequence the interesting end of the PCR product. After filtering for reads that carry a target that is error-free or with a single point mutation, 217,656 and 353,141 reads remained for crR6 and R6 respectively. The relative proportion of each mutant in the crR6 sample over the R6 sample was computed (FIG. 24c). All mutations outside of the seed sequence (13-20 bases away from the PAM) show full interference. Those sequences were used as a reference to determine if other mutations inside the seed sequence can be said to significantly disrupt interference. A normal distribution was fitted to theses sequences using the fitdistr function of the MASS R package. The 0.99 quantile of the fitted distribution is shown as a dotted line in FIG. 24c. FIG. 72 shows a histogram of the data density with fitted normal distribution (black line) and 0.99 quantile (dotted line).

TABLE-US-00011 TABLE F Relative abundance of PAM sequences in the crR6/R6 samples averaged over bases 1 and 5. 3rd 2nd position 4th position A C G T position A AAA 1.04 ACA 1.12 AGA 0.73 ATA 1.10 A AAC 1.07 ACC 1.04 AGC 0.64 ATC 0.97 C AAG 1.00 ACG 1.09 AGG 0.61 ATG 1.07 G AAT 0.98 ACT 1.02 AGT 0.65 ATT 1.01 T C CAA 1.05 CCA 1.05 CGA 0.99 CTA 1.07 A CAC 1.04 CCC 1.02 CGC 1.08 CTC 1.04 C CAG 1.08 CCG 1.08 CGG 0.81 CTG 1.05 G CAT 1.13 CCT 1.05 CGT 1.07 CTT 1.08 T G GAA 0.97 GCA 1.05 GGA 0.08 GTA 0.99 A GAC 0.92 GCC 1.00 GGC 0.05 GTC 1.15 C GAG 0.96 GCG 0.98 GGG 0.07 GTG 0.98 G GAT 0.98 GCT 0.99 GGT 0.06 GTT 1.05 T T TAA 1.08 TCA 1.16 TGA 1.05 TTA 1.14 A TAC 1.00 TCC 1.08 TGC 1.06 TTA 1.05 C TAG 1.02 TCG 1.11 TGG 0.77 TTG 1.01 G TAT 1.01 TCT 1.12 TGT 1.21 TTT 1.02 T

TABLE-US-00012 TABLE G Primers used in this study (SEQ ID NOS 68-183, respectively, in order of appearance). Primer Sequence 5'-3' B217 TCCTAGCAGGATTTCTGATATTACTGTCACGTTTT AGAGCTATGCTGTTTTGA B218 GTGACAGTAATATCAGAAATCCTGCTAGGAGTTTT GGGACCATTCAAAACAGC B229 GGGTTTCAAGTCTTTGTAGCAAGAG B230 GCCAATGAACGGGAACCCTTGGTC B250 NNNNGACGAGGCAATGGCTGAAATC B251 NNNNTTATTTGGCTCATATTTGCTG B255 CTTTACACCAATCGCTGCAACAGAC B256 CAAAATTTCTAGTCTTCTTTGCCTTTCCCCATAAA ACCCTCCTTA B257 AGGGTTTTATGGGGAAAGGCAAAGAAGACTAGAAA TTTTGATACC B258 CTTACGGTGCATAAAGTCAATTTCC B269 TGGCTCGATTTCAGCCATTGC B270 CTTTGACGAGGCAATGGCTGAAATCGAGCCAANAA AGCGCAAG B271 CTTTGACGAGGCAATGGCTGAAATCGAGCCAAANA AGCGCAAG B272 CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAN AGCGCAAG B273 CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAA NGCGCAAG B274 CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAA ANCGCAAG B275 CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAA AGNGCAAG B276 CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAA AGCNCAAGAAG B277 CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAA AGCGNAAGAAG B278 CTTTGACGAGGCAATGGCTGAAATCGAGCCAAAAA AGCGCNAGAAG B279 GCGCTTTTTTGGCTCGATTTCAG B280 CAATGGCTGAAATCGAGCCAAAAAAGCGCANGAAG AAATC B281 CAATGGCTGAAATCGAGCCAAAAAAGCGCAANAAG AAATC B282 CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGNAG AAATC B283 CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGANG AAATC B284 CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAN AAATC B285 CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAG NAATCAACC B286 CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAG ANATCAACC B287 CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAG AANTCAACC B288 CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAG AAANCAACC B289 CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAG AAATNAACCAGC B290 CAATGGCTGAAATCGAGCCAAAAAAGCGCAAGAAG AAATCNACCAGC B296 gatccTCCATCCGTACAACCCACAACCCTGg B297 aattcCAGGGTTGTGGGTTGTACGGATGGAg B298 CATGGATCCTATTTCTTAATAACTAAAAATATGG B299 CATGAATTCAACTCAACAAGTCTCAGTGTGCTG B300 AAACATTTTTTCTCCATTTAGGAAAAAGGATGCTG B301 AAAACAGCATCCTTTTTCCTAAATGGAGAAAAAAT B302 AAACCTTAAATCAGTCACAAATAGCAGCAAAATTG B303 AAAACAATTTTGCTGCTATTTGTGACTGATTTAAG B304 AAACTTTTCATCATACGACCAATCTGCTTTATTTG B305 AAAACAAATAAAGCAGATTGGTCGTATGATGAAAA B306 AAACTCGTCCAGAAGTTATCGTAAAAGAAATCGAG B307 AAAACTCGATTTCTTTTACGATAACTTCTGGACGA B308 AAACAATCTCTCCAAGGTTTCCTTAAAAATCTCTG B309 AAAACAGAGATTTTTAAGGAAACCTTGGAGAGATT B310 AAACGCCATCGTCAGGAAGAAGCTATGCTTGAGTG B311 AAAACACTCAAGCATAGCTTCTTCCTGACGATGGC B312 AAACATCTCTATACTTATTGAAATTTCTTTGTATG B313 AAAACATACAAAGAAATTTCAATAAGTATAGAGAT B314 AAACTAGCTGTGATAGTCCGCAAAACCAGCCTTCG B315 AAAACGAAGGCTGGTTTTGCGGACTATCACAGCTA B316 AAACATCGGAAGGTCGAGCAAGTAATTATCTTTTG B317 AAAACAAAAGATAATTACTTGCTCGACCTTCCGAT B318 AAACAAGATGGTATCGCAAAGTAAGTGACAATAAG B319 AAAACTTATTGTCACTTACTTTGCGATACCATCTT B320 GAGACCTTTGAGCTTCCGAGACTGGTCTCAGTTTT GGGACCATTCAAAACAG B321 TGAGACCAGTCTCGGAAGCTCAAAGGTCTCGTTTT AGAGCTATGCTGTTTTG B352 aaacTACTTTACGCAGCGCGGAGTTCGGTTTTTTg B353 aaaacAAAAAACCGAACTCCGCGCTGCGTAAAGTA HC008_SP ATGCCGGTACTGCCGGGCCTCTTGCGGGATTACGA AATCATCCTG HC009_SP GTGACTGGCGATGCTGTCGGAATGGACGATCACAC TACTCTTCTT HC010_SP TTAAGAAATAATCTTCATCTAAAATATACTTCAGT CACCTCCTAGCTGAC HC011_SP ATTGATTTGAGTCAGCTAGGAGGTGACTGAAGTAT ATTTTAGATGAAG HC014_SP GAGACCTTTGAGCTTCCGAGACTGGTCTCAGTTTT GGGACCATTCAAAACAGCATAGCTCTAAAACCTCG TAGACTATTTTTGTC HC015_SP GAGACCAGTCTCGGAAGCTCAAAGGTCTCGTTTTA GAGCTATGCTGTTTTGAATGGTCCCAAAACTTCAG CACACTGAGACTTG L403 AGTCATCCCAGCAACAAATGG L409 CGTGGTAAATCGGATAACGTTCCAAGTGAAG L422 Tgctcttcttcacaaacaaggg L426 AAGCCAAAGTTTGGCACCACC L430 GTAGCTTATTCAGTCCTAGTGG L444 CGTTTGTTGAACTAATGGGTGCAAATTACGAATCT TCTCCTGACG L445 CGTCAGGAGAAGATTCGTAATTTGCACCCATTAGT TCAACAAACG L446 GATATTATGGAGCCTATTTTTGTGGGTTTTTAGGC ATAAAACTATATG L447 CATATAGTTTTATGCCTAAAAACCcACAAAAATAG GCTCCATAATATC L448 ATTATTTCTTAATAACTAAAAATATGG L457 CGTgtacaattgctagcgtacggc L458 GCACCGGTGATCACTAGTCCTAGG L459 cctaggactagtgatcaccggtGCAAATATGAGCC AAATAAATATAT L461 GCCGTACGCTAGCAATTGTACACGTTTGTTGAACT AATGGGTGC L481 TTCAAATTTTCCCATTTGATTCTCC L488 CCATATTTTTAGTTATTAAGAAATAATACCAGCCA TCAGTCACCTCC W256 AGACGATTCAATAGACAATAAGG W286 GTTTTGGGACCATTCAAAACAGCATAGCTCTAAAA CCTCGTAGAC W287 GCTATGCTGTTTTGAATGGTCCCAAAACcattatt ttaacacacgaggtg W288 GCTATGCTGTTTTGAATGGTCCCAAAACGCACCCA TTAGTTCAACAAACG W326 AATTCTTTTCTTCATCATCGGTC W327 AAGAAAGAATGAAGATTGTTCATG W341 GGTACTAATCAAAATAGTGAGGAGG W354 GTTTTTCAAAATCTGCGGTTGCG W355 AAAAATTGAAAAAATGGTGGAAACAC W356 ATTTCGTAAACGGTATCGGTTTCTTTTAAAGTTTT GGGACCATTCAAAACAGC W357 TTTAAAAGAAACCGATACCGTTTACGAAATGTTTT AGAGCTATGCTGTTTTGA W365 AAACGGTATCGGTTTCTTTTAAATTCAATTGTTTT GGGACCATTCAAAACAGC W366 AATTCAATTTAAAAGAAACCGATACCGTTTGTTTT AGAGCTATGCTGTTTTGA W370 GTTCCTTAAACCAAAACGGTATCGGTTTCTTTTAA ATTC W371 GAAACCGATACCGTTTTGGTTTAAGGAACAGGTAA AGGGCATTTAAC

W376 CGATTTCAGCCATTGCCTCGTC W377 GCCTTTGACGAGGCAATGGCTGAAATCGNNNNNAA AAAGCGCAAGAAGAAATCAAC W391 TCCGTACAACCCACAACCCTGCTAGTGAGCGTTTT GGGACCATTCAAAACAGC W392 GCTCACTAGCAGGGTTGTGGGTTGTACGGAGTTTT AGAGCTATGCTGTTTTGA W393 TTGTTGCCACTCTTCCTTCTTTC W397 CAGGGTTGTGGGTTGTTGCGATGGAGTTAACTCCC ATCTCC W398 GGGAGTTAACTCCATCGCAACAACCCACAACCCTG CTAGTG W403 GTGGTATCTATCGTGATGTGAC W404 TTACCGAAACGGAATTTATCTGC W405 AAAGCTAGAGTTCCGCAATTGG W431 GTGGGTTGTACGGATTGAGTTAACTCCCATCTCCT TC W432 GATGGGAGTTAACTCAATCCGTACAACCCACAACC CTG W433 GCTTCACCTATTGCAGCACCAATTGACCACATGAA GATAG W434 GTGGTCAATTGGTGCTGCAATAGGTGAAGCTAATG GTGATG W463 CTGATTTGTATTAATTTTGAGACATTATGCTTCAC CTTC W464 GCATAATGTCTCAAAATTAATACAAATCAGTGAAA TCATG W465 GTTTTGGGACCATTCAAAACAGCATAGCTCTAAAA CGTGACAGTAATATCAG W466 GTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAA CGCTCACTAGCAGGGTTG W542 ATACTTTACGCAGCGCGGAGTTCGGTTTTgTAGGA GTGGTAGTATATACACGAGTACAT

TABLE-US-00013 TABLE H Design of targeting and editing constructs used in this study (SEQ ID NOS 184, 184, 184, 185, and 186, respectively, in order of appearance). Targeting Constructs Edition Template DNA Left PCR Right PCR Spacer sequence PAM bgaA R > A crR6Rk W256/W391 W392/L403 GCTCACTAGCAGGGT TGG TGTGGGTTGTACGGA bgaA NE > AA crR6Rk W256/W391 W392/L403 GCTCACTAGCAGGGT TGG TGTGGGTTGTACGGA .DELTA.bgaA crR6Rk W256/W391 W392/L403 GCTCACTAGCAGGGT TGG TGTGGGTTGTACGGA .DELTA.srtA crR6Rc W256/B218 B217/L403 TCCTAGCAGGATTTC TGG TGATATTACTGTCAC ermB Stop crR6Rk W256/W356 W357/L403 TTTAAAAGAAACCGA TGG TACCGTTTACGAAAT .DELTA.srtA .DELTA.bgaA JEN51 W256/W465 W466/W403 same as the TGG (for Left PCR) ones used for and .DELTA.srtA and .DELTA.bgaA JEN52 (for Right PCR) Editing Constructs Primers used Name of to verify Template SOEing resulting edited Edition DNA PCR A PCR B PCR C PCR strains genotype bgaA R > A R6 W403/W397 W398/W404 N/A W403/W404 JEN55 W403/W404 bgaA NE > AA R6 W403/W431 W432/W433 W434/W404 W403/W404 JEN60 W403/W404 .DELTA.bgaA R6 B255/B256 B257/B258 N/A B255/B258 JEN52 W393/W405 .DELTA.srtA R6 B230/W463 W464/B229 N/A B230/B229 JEN51 W422/W426 ermB Stop JEN38 L122/W370 W371/L426 N/A L422/L426 JEN43 L457/L458 .DELTA.srtA .DELTA.bgaA same as the JEN64 same as the ones used ones used for .DELTA.srtA for .DELTA.srtA and .DELTA.bgaA and .DELTA.bgaA

Example 6

Optimization of the Guide RNA for Streptococcus pyogenes Cas9 (Referred to as SpCas9)

[0304] Applicants mutated the tracrRNA and direct repeat sequences, or mutated the chimeric guide RNA to enhance the RNAs in cells.

[0305] 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.

TABLE-US-00014 Optimized tracrRNA 1 (mutation underlined): (SEQ ID NO: 187) GGAACCATTCAtAACAGCATAGCAAGTTAtAATAAGGCTAGTCCGTTA TCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT Optimized direct repeat 1 (mutation underlined): (SEQ ID NO: 188) GTTaTAGAGCTATGCTGTTaTGAATGGTCCCAAAAC Optimized tracrRNA 2 (mutation underlined): (SEQ ID NO: 189) GGAACCATTCAAtACAGCATACGCAAGTTAAtATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT Optimized direct repeat 2 (mutation underlined): (SEQ ID NO: 190) GTaTTAGAGCTATGCTGTaTTGAATGGTCCCAAAAC

[0306] Applicants also optimized the chimetic guideRNA for optimal activity in eukaryotic cells.

TABLE-US-00015 Original guide RNA: (SEQ ID NO: 191) NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGCTTTTTTT Optimized chimeric guide RNA sequence 1: (SEQ ID NO: 192) NNNNNNNNNNNNNNNNNNNNGTATTAGAGCTAGAAATAGC AAGTTAATATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGCTTTTTTT Optimized chimeric guide RNA sequence 2: (SEQ ID NO: 193) NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTATGGTGTTT TGGAAACAAAACAGCATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTT Optimized chimeric guide RNA sequence 3: (SEQ ID NO: 194) NNNNNNNNNNNNNNNNNNNNGTATTAGAGCTATGCTGTAT TGGAAACAATACAGCATAGCAAGTTAATATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTT

[0307] Applicants showed that optimized chimeric guide RNA works better as indicated in FIG. 3. 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 Emx1 locus: "GTCACCTCCAATGACTAGGG (SEQ ID NO: 195)"

Example 7

Optimization of Streptococcus thermophiles LMD-9 CRISPR1 Cas9 (Referred to as StlCas9)

[0308] Applicants designed guide chimeric RNAs as shown in FIG. 4.

[0309] The StlCas9 guide RNAs can undergo the same type of optimization as for SpCas9 guide RNAs, by breaking the stretches of poly thymines (Ts)

Example 8

Cas9 Diversity and Mutations

[0310] The CRISPR-Cas system is an adaptive immune mechanism against invading exogenous DNA employed by diverse species across bacteria and archaea. The type II CRISPR-Cas9 system consists of a set of genes encoding proteins responsible for the "acquisition" of foreign DNA into the CRISPR locus, as well as a set of genes encoding the "execution" of the DNA cleavage mechanism; these include the DNA nuclease (Cas9), a non-coding transactivating cr-RNA (tracrRNA), and an array of foreign DNA-derived spacers flanked by direct repeats (crRNAs). Upon maturation by Cas9, the tracRNA and crRNA duplex guide the Cas9 nuclease to a target DNA sequence specified by the spacer guide sequences, and mediates double-stranded breaks in the DNA near a short sequence motif in the target DNA that is required for cleavage and specific to each CRISPR-Cas system. The type II CRISPR-Cas systems are found throughout the bacterial kingdom and highly diverse in in Cas9 protein sequence and size, tracrRNA and crRNA direct repeat sequence, genome organization of these elements, and the motif requirement for target cleavage. One species may have multiple distinct CRISPR-Cas systems.

[0311] Applicants evaluated 207 putative Cas9s from bacterial species identified based on sequence homology to known Cas9s and structures orthologous to known subdomains, including the HNH endonuclease domain and the RuvC endonuclease domains [information from the Eugene Koonin and Kira Makarova]. Phylogenetic analysis based on the protein sequence conservation of this set revealed five families of Cas9s, including three groups of large Cas9s (.about.1400 amino acids) and two of small Cas9s (.about.1100 amino acids) (FIGS. 39 and 40A-F).

[0312] In this example, Applicants show that the following mutations can convert SpCas9 into a nicking enzyme: D10A, E762A, H840A, N854A, N863A, D986A.

[0313] Applicants provide sequences showing where the mutation points are located within the SpCas9 gene (FIG. 41). Applicants also show that the nickases are still able to mediate homologous recombination (Assay indicated in FIG. 2). Furthermore, Applicants show that SpCas9 with these mutations (individually) do not induce double strand break (FIG. 47).

Example 9

Supplement to DNA Targeting Specificity of the RNA-Guided Cas9 Nuclease

[0314] Cell Culture and Transfection

[0315] 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), 100 U/mL penicillin, and 100 U/mL streptomycin at 37.degree. C. with 5% CO2 incubation.

[0316] 293FT cells were seeded either onto 6-well plates, 24-well plates, or 96-well plates (Corning) 24 hours prior to transfection. Cells were transfected using Lipofectamine 2000 (Life Technologies) at 80-90% confluence following the manufacturer's recommended protocol. For each well of a 6-well plate, a total of 1 ug of Cas9+sgRNA plasmid was used. For each well of a 24-well plate, a total of 500 ng Cas9+sgRNA plasmid was used unless otherwise indicated. For each well of a 96-well plate, 65 ng of Cas9 plasmid was used at a 1:1 molar ratio to the U6-sgRNA PCR product.

[0317] Human embryonic stem cell line HUES9 (Harvard Stem Cell Institute core) was maintained in feeder-free conditions on GelTrex (Life Technologies) in mTesR medium (Stemcell Technologies) supplemented with 100 ug/ml Normocin (InvivoGen). HUES9 cells were transfected with Amaxa P3 Primary Cell 4-D Nucleofector Kit (Lonza) following the manufacturer's protocol.

[0318] SURVEYOR Nuclease Assay for Genome Modification

[0319] 293FT cells were transfected with plasmid DNA as described above. Cells were incubated at 37.degree. 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.degree. C. for 15 minutes and 98.degree. C. for 10 minutes.

[0320] The genomic region flanking the CRISPR target site for each gene was PCR amplified (primers listed in Tables J and K), 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 .mu.l 10.times.Taq DNA Polymerase PCR buffer (Enzymatics) and ultrapure water to a final volume of 2011, and subjected to a re-annealing process to enable heteroduplex formation: 95.degree. C. for 10 min, 95.degree. C. to 85.degree. C. ramping at -2.degree. C./s, 85.degree. C. to 25.degree. C. at -0.25.degree. C./s, and 25.degree. 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.

[0321] Northern Blot Analysis of tracrRNA Expression in Human Cells

[0322] Northern blots were performed as previously described 1. Briefly, RNAs were heated to 95.degree. C. for 5 min before loading on 8% denaturing polyacrylamide gels (SequaGel, National Diagnostics). Afterwards, RNA was transferred to a pre-hybridized Hybond N+ membrane (GE Healthcare) and crosslinked with Stratagene UV Crosslinker (Stratagene). Probes were labeled with [gamma-32P]ATP (Perkin Elmer) with T4 polynucleotide kinase (New England Biolabs). After washing, membrane was exposed to phosphor screen for one hour and scanned with phosphorimager (Typhoon).

[0323] Bisulfite Sequencing to Assess DNA Methylation Status

[0324] HEK 293FT cells were transfected with Cas9 as described above. Genomic DNA was isolated with the DNeasy Blood & Tissue Kit (Qiagen) and bisulfite converted with EZ DNA Methylation-Lightning Kit (Zymo Research). Bisulfite PCR was conducted using KAPA2G Robust HotStart DNA Polymerase (KAPA Biosystems) with primers designed using the Bisulfite Primer Seeker (Zymo Research, Tables J and K). Resulting PCR amplicons were gel-purified, digested with EcoRI and HindII, and ligated into a pUC19 backbone prior to transformation. Individual clones were then Sanger sequenced to assess DNA methylation status.

[0325] In Vitro Transcription and Cleavage Assay

[0326] HEK 293FT cells were transfected with Cas9 as described above. Whole cell lysates were then prepared with a lysis buffer (20 mM HEPES, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 0.1% Triton X-100) supplemented with Protease Inhibitor Cocktail (Roche). T7-driven sgRNA was in vitro transcribed using custom oligos (Example 10) and HiScribe T7 In Vitro Transcription Kit (NEB), following the manufacturer's recommended protocol. To prepare methylated target sites, pUC19 plasmid was methylated by M.SssI and then linearized by NheI. The in vitro cleavage assay was performed as follows: for a 20 uL cleavage reaction, 10 uL of cell lysate with incubated with 2 uL cleavage buffer (100 mM HEPES, 500 mM KCl, 25 mM MgCl2, 5 mM DTT, 25% glycerol), the in vitro transcribed RNA, and 300 ng pUC19 plasmid DNA.

[0327] Deep Sequencing to Assess Targeting Specificity

[0328] HEK 293FT cells plated in 96-well plates were transfected with Cas9 plasmid DNA and single guide RNA (sgRNA) PCR cassette 72 hours prior to genomic DNA extraction (FIG. 72). The genomic region flanking the CRISPR target site for each gene was amplified (FIG. 74, FIG. 80, (Example 10) by a fusion PCR method to attach the Illumina P5 adapters as well as unique sample-specific barcodes to the target amplicons (schematic described in FIG. 73). PCR products were purified using EconoSpin 96-well Filter Plates (Epoch Life Sciences) following the manufacturer's recommended protocol.

[0329] Barcoded and purified DNA samples were quantified by Quant-iT PicoGreen dsDNA Assay Kit or Qubit 2.0 Fluorometer (Life Technologies) and pooled in an equimolar ratio. Sequencing libraries were then deep sequenced with the Illumina MiSeq Personal Sequencer (Life Technologies).

[0330] Sequencing Data Analysis and Indel Detection

[0331] MiSeq reads were filtered by requiring an average Phred quality (Q score) of at least 23, as well as perfect sequence matches to barcodes and amplicon forward primers. Reads from on- and off-target loci were analyzed by first performing Smith-Waterman alignments against amplicon sequences that included 50 nucleotides upstream and downstream of the target site (a total of 120 bp). Alignments, meanwhile, were analyzed for indels from 5 nucleotides upstream to 5 nucleotides downstream of the target site (a total of 30 bp). Analyzed target regions were discarded if part of their alignment fell outside the MiSeq read itself, or if matched base-pairs comprised less than 85% of their total length.

[0332] Negative controls for each sample provided a gauge for the inclusion or exclusion of indels as putative cutting events. For each sample, an indel was counted only if its quality score exceeded .mu.-.sigma., where .mu. was the mean quality-score of the negative control corresponding to that sample and .sigma. was the standard deviation of same. This yielded whole target-region indel rates for both negative controls and their corresponding samples. Using the negative control's per-target-region-per-read error rate, q, the sample's observed indel count n, and its read-count R, a maximum-likelihood estimate for the fraction of reads having target-regions with true-indel s, P, was derived by applying a binomial error model, as follows.

[0333] Letting the (unknown) number of reads in a sample having target regions incorrectly counted as having at least 1 indel be E, we can write without making any assumptions about the number of true indels)

Prob ( E p ) = ( R ( 1 - p ) E ) q E ( 1 - q ) R ( 1 - p ) - E ##EQU00001##

[0334] since R(1-p) is the number of reads having target-regions with no true indels. Meanwhile, because the number of reads observed to have indels is n, n=E+Rp, in other words the number of reads having target-regions with errors but no true indels plus the number of reads whose target-regions correctly have indels. We can then re-write the above

Prob ( E p ) = Prob ( n = E + Rp p ) = ( R ( 1 - p ) n - Rp ) q n - Rp ( 1 - q ) R - n ##EQU00002##

[0335] Taking all values of the frequency of target-regions with true-indels P to be equally probable a priori, Prob(n|P).varies.Prob(p|n). The maximum-likelihood estimate (MLE) for the frequency of target regions with true-indels was therefore set as the value of P that maximized Prob(n|p). This was evaluated numerically.

[0336] In order to place error bounds on the true-indel read frequencies in the sequencing libraries themselves, Wilson score intervals (2) were calculated for each sample, given the MLE-estimate for true-indel target-regions, Rp, and the number of reads R. Explicitly, the lower bound l and upper bound u were calculated as

l = ( Rp + z 2 2 - z Rp ( 1 - p ) + z 2 / 4 ) / ( R + z 2 ) ##EQU00003## u = ( Rp + z 2 2 + z Rp ( 1 - p ) + z 2 / 4 ) / ( R + z 2 ) ##EQU00003.2##

[0337] where z, the standard score for the confidence required in normal distribution of variance 1, was set to 1.96, meaning a confidence of 95%. The maximum upper bounds and minimum lower bounds for each biological replicate are listed in FIGS. 80-83.

[0338] qRT-PCR Analysis of Relative Cas9 and sgRNA Expression

[0339] 293FT cells plated in 24-well plates were transfected as described above. 72 hours post-transfection, total RNA was harvested with miRNeasy Micro Kit (Qiagen). Reverse-strand. synthesis for sgRNAs was performed with qScript Flex cDNA kit (MR) and custom first-strand synthesis primers (Tables J and K). qPCR analysis was performed with Fast SYBR Green Master Mix (Life Technologies) and custom primers (Tables J and K), using GAPDH as an endogenous control. Relative quantification was calculated by the .DELTA..DELTA.CT method.

TABLE-US-00016 TABLE I Target site sequences. Tested target sites for S. pyogenes type II CRISPR system with the requisite PAM. Cells were transfected with Cas9 and either crRNA-tracrRNA or chimeric sgRNA for each target. Target genomic Target site site ID target sequence (5' to 3') PAM strand 1 EMX1 GTCACCTCCAATGACTAGGG TGG + (SEQ ID NO: 319) 2 EMX1 GACATCGATGTCCTCCCCAT TGG - (SEQ ID NO: 196) 3 EMX1 GAGTCCGAGCAGAAGAAGAA GGG + (SEQ ID NO: 197) 6 EMX1 GCGCCACCGGTTGATGTGAT GGG - (SEQ ID NO: 198) 10 EMX1 GGGGCACAGATGAGAAACTC AGG - (SEQ ID NO: 199) 11 EMX1 GTACAAACGGCAGAAGCTGG AGG + (SEQ ID NO: 200) 12 EMX1 GGCAGAAGCTGGAGGAGGAA GGG + (SEQ ID NO: 201) 13 EMX1 GGAGCCCTTCTTCTTCTGCT CGG - (SEQ ID NO: 202) 14 EMX1 GGGCAACCACAAACCCACGA GGG + (SEQ ID NO: 203) 15 EMX1 GCTCCCATCACATCAACCGG TGG + (SEQ ID NO: 204) 16 EMX1 GTGGCGCATTGCCACGAAGC AGG + (SEQ ID NO: 205) 17 EMX1 GGCAGAGTGCTGCTTGCTGC TGG + (SEQ ID NO: 206) 18 EMX1 GCCCCTGCGTGGGCCCAAGC TGG + (SEQ ID NO: 207) 19 EMX1 GAGTGGCCAGAGTCCAGCTT GGG - (SEQ ID NO: 208) 20 EMX1 GGCCTCCCCAAAGCCTGGCC AGG - (SEQ ID NO: 209) 4 PVALB GGGGCCGAGATTGGGTGTTC AGG + (SEQ ID NO: 210) 5 PVALB GTGGCGAGAGGGGCCGAGAT TGG + (SEQ ID NO: 211) 1 SERPINB5 GAGTGCCGCCGAGGCGGGGC GGG + (SEQ ID NO: 212) 2 SERPINB5 GGAGTGCCGCCGAGGCGGGG CGG + (SEQ ID NO: 213) 3 SERPINB5 GGAGAGGAGTGCCGCCGAGG CGG + (SEQ ID NO: 214)

TABLE-US-00017 TABLE J Primer sequences SURVEYOR assay primer genomic primer sequence name target (5' to 3') Sp-EMX1-F1 EMX1 AAAACCACCCTTCTCTCTGGC (SEQ ID NO: 36) Sp-EMX1-R1 EMX1 GGAGATTGGAGACACGGAGAG (SEQ ID NO: 37) Sp-EMX1-F2 EMX1 CCATCCCCTTCTGTGAATGT (SEQ ID NO: 215) Sp-EMX1-R2 EMX1 GGAGATTGGAGACACGGAGA (SEQ ID NO: 216) Sp-PVALB-F PVALB CTGGAAAGCCAATGCCTGAC (SEQ ID NO: 38) Sp-PVALB-R PVALB GGCAGCAAACTCCTTGTCCT (SEQ ID NO: 39) qRT-PCR for Cas9 and sgRNA expression primer primer sequence name (5' to 3') sgRNA reverse- AAGCACCGACTCGGTGCCAC strand synthesis (SEQ ID NO: 217) EXM1.1 sgRNA TCACCTCCAATGACTAGGGG qPCR F (SEQ ID NO: 218) EXM1.1 sgRNA CAAGTTGATAACGGACTAGCCT qPCR R (SEQ ID NO: 219) EXM1.3 sgRNA AGTCCGAGCAGAAGAAGAAGTTT qPCR F (SEQ ID NO: 220) EXM1.3 sgRNA TTTCAAGTTGATAACGGACTAGCCT qPCR R (SEQ ID NO: 221) Cas9 qPCR F AAACAGCAGATTCGCCTGGA (SEQ ID NO: 222) Cas9 qPCR R TCATCCGCTCGATGAAGCTC (SEQ ID NO: 223) GAPDH qPCR F TCCAAAATCAAGTGGGGCGA (SEQ ID NO: 224) GAPDH qPCR R TGATGACCCTTTTGGCTCCC (SEQ ID NO: 225) Bisulfate PCR and sequencing primer primer sequence name (5' to 3') Bisulfate PCR F GAGGAATTCTTTTTTTGTTYGAATA (SERPINB5 locus) TGTTGGAGGTTTTTTGGAAG (SEQ ID NO: 226) Bisulfate PCR R GAGAAGCTTAAATAAAAAACRACAA (SERPINB5 locus) TACTCAACCCAACAACC (SEQ ID NO: 227) pUC19 sequencing CAGGAAACAGCTATGAC (SEQ ID NO: 228)

TABLE-US-00018 TABLE K Sequences for primers to test sgRNA architecture. Primers hybridize to the reverse strand of the U6 promoter unless otherwise indicated. The U6 priming site is in italics, the guide sequence is indicated as a stretch of Ns, the direct repeat sequence is highlighted in bold, and the tracrRNA sequence underlined. The secondary structure of each sgRNA architecture is shown in FIG. 43. primer name primer sequence (5' to 3') U6-Forward GCCTCTAGAGGTACCTGAGGGCCTAT TTCCCATGATTCC (SEQ ID NO: 229) I: sgRNA(DR +12, ACCTCTAGAAAAAAAGCACCGACTCG tracrRNA +85) GTGCCACTTTTTCAAGTTGATAACGG ACTAGCCTTATTTTAACTTGCTATTT CTAGCTCTAAAACNNNNNNNNNNNNN NNNNNNNGGTGTTTTCGTCCTTTCCA CAAG (SEQ ID NO: 230) II: sgRNA(DR +12, ACCTCTAGAAAAAAAGCACCGACTCG tracrRNA +85) GTGCCACTTTTTCAAGTTGATAACGG mut2 ACTAGCCTTATATTAACTTGCTATTT CTAGCTCTAATACNNNNNNNNNNNNN NNNNNNNGGTGTTTCGTCCTTTCCAC AAG (SEQ ID NO: 231) III: sgRNA(DR +22, ACCTCTAGAAAAAAAGCACCGACTTC tracrRNA +85) GGTGCCACTTTTTCAAGTTGATAACG GACTAGCCTTATTTTAACTTGCTATG CTGTTTTGTTTCCAAAACAGCATAGC TCTAAAACNNNNNNNNNNNNNNNNNN NNGGTGTTTCGTCCTTTCCACAAG (SEQ ID NO: 232) IV: sgRNA(DR +22, ACCTCTAGAAAAAAAGCACCGACTTC tracrRNA +85) GGTGCCACTTTTTCAAGTTGATAACG mut4 GACTAGCCTTATATTAACTTGCTATG CTGTATTGTTTCCAATACAGCATAGC TCTAATACNNNNNNNNNNNNNNNNNN NNGGTGTTTCGTCCTTTCCACAAG (SEQ ID NO: 233)

TABLE-US-00019 TABLE L Target sites with alternate PAMs for testing PAM specificity of Cas9, All target sites for PAM specificity testing are found within the human EMX1 locus. Target site sequence (5' to 3') PAM AGGCCCCAGTGGCTGCTCT (SEQ ID NO: 234) NAA ACATCAACCGGTGGCGCAT (SEQ ID NO: 235) NAT AAGGTGTGGTTCCAGAACC (SEQ ID NO: 236) NAC CCATCACATCAACCGGTGG (SEQ ID NO: 237) NAG AAACGGCAGAAGCTGGAGG (SEQ ID NO: 238) NTA GGCAGAAGCTGGAGGAGGA (SEQ ID NO: 239) NTT GGTGTGGTTCCAGAACCGG (SEQ ID NO: 240) NTC AACCGGAGGACAAAGTACA (SEQ ID NO: 241) NTG TTCCAGAACCGGAGGACAA (SEQ ID NO: 242) NCA GTGTGGTTCCAGAACCGGA (SEQ ID NO: 243) NCT TCCAGAACCGGAGGACAAA (SEQ ID NO: 244) NCC CAGAAGCTGGAGGAGGAAG (SEQ ID NO: 245) NCG CATCAACCGGTGGCGCATT (SEQ ID NO: 246) NGA GCAGAAGCTGGAGGAGGAA (SEQ ID NO: 247) NGT CCTCCCTCCCTGGCCCAGG (SEQ ID NO: 248) NGC TCATCTGTGCCCCTCCCTC (SEQ ID NO: 249) NAA GGGAGGACATCGATGTCAC (SEQ ID NO: 250) NAT CAAACGGCAGAAGCTGGAG (SEQ ID NO: 251) NAC GGGTGGGCAACCACAAACC (SEQ ID NO: 252) NAG GGTGGGCAACCACAAACCC (SEQ ID NO: 253) NTA GGCTCCCATCACATCAACC (SEQ ID NO: 254) NTT GAAGGGCCTGAGTCCGAGC (SEQ ID NO: 255) NTC CAACCGGTGGCGCATTGCC (SEQ ID NO: 256) NTG AGGAGGAAGGGCCTGAGTC (SEQ ID NO: 257) NCA AGCTGGAGGAGGAAGGGCC (SEQ ID NO: 258) NCT GCATTGCCACGAAGCAGGC (SEQ ID NO: 259) NCC ATTGCCACGAAGCAGGCCA (SEQ ID NO: 260) NCG AGAACCGGAGGACAAAGTA (SEQ ID NO: 261) NGA TCAACCGGTGGCGCATTGC (SEQ ID NO: 262) NGT GAAGCTGGAGGAGGAAGGG (SEQ ID NO: 263) NGC

Example 10

Supplementary Sequences

[0340] All sequences are in the 5' to 3' direction. For U6 transcription, the string of underlined Ts serve as the transcriptional terminator.

TABLE-US-00020 > U6-short tracrRNA (Streptococcus pyogenes SF370) (SEQ ID NO: 40) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa- tttgactgtaaa cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat- gttttaaaatggactatcatatgc ttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccGGAACCATT- CAAAACAGC ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA GTCGGTGCTTTTTTT (tracrRNA sequence in bold) >U6-DR-guide sequence-DR (Streptococcus pyogenes SF370) (SEQ ID NO: 54) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa- tttgactgtaaa cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtagcagtataaaattatga- ttaaaatggactatcatatgc ##STR00001## ##STR00002## TTTTTT ##STR00003## > sgRNA containing +48 tracrRNA (Streptococcus pyogenes SF370) (SEQ ID NO: 55) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa- tttgactgtaaa cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat- gtataaaatggactatcatatgc ttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNN- NNNNNNNNN ##STR00004## (guide sequence is in bold Ns and the tracrRNA fragment is in bold) > sgRNA containing +54 tracrRNA (Streptococcus pyogenes SF370) (SEQ ID NO: 56) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgnagagagataattggaataatt- tgactgtaaa cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat- gtataaaatggactatcatatgc ttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNN- NNNNNNNNN ##STR00005## (guide sequence is in bold Ns and the tracrRNA fragment is in bold) > sgRNA containing +67 tracrRNA (Streptococcus pyogenes SF370) (SEQ ID NO: 57) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa- tttgactgtaaa cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtagcagtataaaattatgt- ataaaatggactatcatatgc ttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNN- NNNNNNNNN ##STR00006## (guide sequence is in bold Ns and the tracrRNA fragment is in bold) > sgRNA containing +85 tracrRNA (Streptococcus pyogenes SF370) (SEQ ID NO: 58) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaa- tttgactgtaaa cacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtagcagtataaaattatga- ttaaaatggactatcatatgc ttaccgtaacttgaaagtatttcgatttcttggattatatatcttgtggaaaggacgaaacaccNNNNNNNNNN- NNNNNNNN ##STR00007## T (guide sequence is in bold Ns and the tracrRNA fragment is in bold) > CBh-NLS-SpCas9-NLS (SEQ ID NO: 59) CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTT TCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA CGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTC CCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTG TGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCG GCGCGCTCCGAAAGTTTCCTTCTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAA AAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCC GCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCAC AGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGG TAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCAC CTGCCTGAAATCACTTTTTTTCAGGTTGGaccggtgccaccATGGACTATAAGGACCACG ACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATG GCCCCAAAGAAGAAGCGGAAGGTVGGTATCCACGGAGTCCCAGCAGCCGACAA GAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGA TCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACC GACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGG CGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCA GACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCC AAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGA GGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGG CCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGAC AGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGAT CAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCG ACGTGGACAAGCTGTTCATCCAGGTGGTGCAGACCTACAACCAGCTGTTCGAG GAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAG ACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGA AGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCC AACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAA GGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGT ACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGC GACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTAT GATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCG TGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAG AACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAA GTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGA AGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAG CATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGG AAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTG ACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATT CGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGG AAGTGGTGGACAAGGCCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAAC TTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGG GAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGAC CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTA CTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATC GGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGAC AAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCT GACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCT ATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATAC ACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGC AGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGA AACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCA GAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATC TGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACACTGAAGGTGGTG GACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGA AATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAG AGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAA AGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACT ACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGG CTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGA CTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCG ACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAG CTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGC CGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCG GATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGA TCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACA AAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCC GTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGT GTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGC AGGAAATCGGCAAGCCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACT TTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTG ATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATT TTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAG ACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAA CAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCG GCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAA AAGGGCAAGTCCAAGAAACTGAAGAGTGTGANAGAGCTGCTGGGGATCACCAT

CATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGG GCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTG TTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCA GAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGG CCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAG CTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAG CGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGT CCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATC ATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTT GACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGC CACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGT CTCAGCTGGGAGGCGACTTTCTTTTTCTTAGCTTGACCAGCTTTCTTAGTAGCA GCAGGACGCTTTAA (NLS-hSpCas9-NLS is highlighted in bold) > Sequencing amplicon for EMX1 guides 1.1, 1.14, 1.17 (SEQ ID NO: 264) CCAATGGGGAGGACATCGATGTCACCTCCAATGACTAGGGTGGGCAACC ACAAACCCACGAGGGCAGAGTGCTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCCC AAGCTGGACTCTGGCCAC > Sequencing amplicon for EMX1 guides 1.2, 1.16 (SEQ ID NO: 265) CGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCC ACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACTAGGGTGGGCA ACCACAAACCCACGAG > Sequencing amplicon for EMX1 guides 1.3, 1.13, 1,15 (SEQ ID NO: 266) GGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTC CGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGC AGGCCAATGGGGAGGACATCGAT > Sequencing amplicon for EMX1 guides 1.6 (SEQ ID NO: 267) AGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTC CCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCG ATGTCACCTCCAATGACTAGGGTGG > Sequencing amplicon for EMX1 guides 1.10 (SEQ ID NO: 268) CCTCAGTCTTCCCATCAGGCTCTCAGCTCAGCCTGAGTGTTGAGGCCCCAG TGGCTGCTCTGGGGGCCTCCTGAGTTTCTCATCTGTGCCCCTCCCTCCCTGGCCCAGG TGAAGGTGTGGTTCCA > Sequencing amplicon for EMX1 guides 1.11, 1.12 (SEQ ID NO: 269) TCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCCAGAACC GGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCA GAAGAAGAAGGGCTCCCATCACA > Sequencing amplicon for EMX1 guides 1.18, 1.19 (SEQ ID NO: 270) CTCCAATGACTAGGGTGGGCAACCACAAACCCACGAGGGCAGAGTGCTG CTTGCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCCACTCCCTGGC CAGGCTTTGGGGAGGCCTTGGAGT > Sequencing amplicon for EMX1 guides 1.20 (SEQ ID NO: 271) CTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCC ACTCCCTGGCCAGGCTTTGGGGAGGCCTGGAGTCATGGCCCCACAGGGCTTGAAGC CCGGGGCCGCCATTGACAGAG >T7 promoter F primer for annealing with target strand (SEQ ID NO: 272) GAAATTAATACGACTCACTATAGGG >oligo containing pUC19 target site 1 for methylation (T7 reverse) (SEQ ID NO: 273) AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCC TTATTTTAACTTGCTATTTCTAGCTCTAAAACAACGACGAGCGTGACACCACCCTAT AGTGAGTCGTATTAATTTC >oligo containing pUC19 target site 2 for methylation (T7 reverse) (SEQ ID NO: 274) AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCC TTATTTTAACTTGCTATTTCTAGCTCTAAAACGCAACAATTAATAGACTGGACCTATA GTGAGTCGTATTAATTTC

Example 11

Oligo-Mediated Cas9-Induced Homologous Recombination

[0341] The oligo homologous recombination test is a comparison of efficiency across different Cas9 variants and different HR template (oligo vs. plasmid).

[0342] 293FT cells were used. SpCas9=Wildtype Cas9 and SpCas9n=nickase Cas9 (D10A). The chimeric RNA target is the same EMAI Protospacer Target 1 as in Examples 5, 9 and 10 and oligos synthesized by IDT using PAGE purification.

[0343] FIG. 44 depicts a design of the oligo DNA used as Homologous Recombination (HR) template in this experiment. Long oligos contain 100 bp homology to the EMX1 locus and a HindIII restriction site. 293FT cells were co-transfected with: first, a plasmid containing a chimeric RNA targeting human EMX1 locus and wild-type cas9 protein, and second, the oligo DNA as HR template. Samples are from 293FT cells collected 96 hours post transfection with Lipofectamine 2000. All products were amplified with an EMX1 HR Primer, gel purified, followed by digestion with HindIII to detect the efficiency of integration of HR template into the human genome.

[0344] FIGS. 45 and 46 depict a comparison of HR efficiency induced by different combination of Cas9 protein and HR template. The Cas9 construct used were either wild-type Cas9 or the nickase version of Cas9 (Cas9n). The HR template used were: antisense oligo DNA (Antisense-Oligo in above figure), or sense oligo DNA (Sense-Oligo in above figure), or plasmid HR template (HR template in above figure). The sense/anti-sense definition is that the actively-transcribed strand with sequence corresponding to the transcribed mRNA is defined as the sense strand of genome. HR Efficiency is shown as percentage of HindIII digestion band as against all genomic PCR amplified product (bottom numbers).

Example 12

Autistic Alouse

[0345] Recent large-scale sequencing initiatives have produced a large number of genes associated with disease. Discovering the genes is only the beginning in understanding what the gene does and how it leads to a diseased phenotype. Current technologies and approaches to study candidate genes are slow and laborious. The gold standards, gene targeting and genetic knockouts, require a significant investment in time and resources, both monetary and in terms of research personnel. Applicants set out to utilize the hSpCas9 nuclease to target many genes and do so with higher efficiency and lower turnaround compared to any other technology. Because of the high efficiency of hSpCas9 Applicants can do RNA injection into mouse zygotes and immediately get genome-modified animals without the need to do any preliminary gene targeting in mESCs.

[0346] Chromodomain helicase DNA binding protein 8 (CHD8) is a pivotal gene in involved in early vertebrate development and morphogenesis. Mice lacking CHD8 die during embryonic development. Mutations in the CHD8 gene have been associated with autism spectrum disorder in humans. This association was made in three different papers published simultaneously in Nature. The same three studies identified a plethora of genes associated with autism spectrum disorder. Applicants' aim was to create knockout mice for the four genes that were found in all papers, Chd8, Katna12, Kctd13, and Scn2a. In addition, Applicants chose two other genes associated with autism spectrum disorder, schizophrenia, and ADHD, GIT1, CACNA1C, and CACNB2. And finally, as a positive control Applicants decide to target MeCP2.

[0347] For each gene Applicants designed three gRNAs that would likely knockout the gene. A knockout would occur after the hSpCas9 nuclease makes a double strand break and the error prone DNA repair pathway, non-homologous end joining, corrects the break, creating a mutation. The most likely result is a frameshift mutation that would knockout the gene. The targeting strategy involved finding proto-spacers in the exons of the gene that had a PAM sequence, NGG, and was unique in the genome. Preference was given to proto-spacers in the first exon, which would be most deleterious to the gene.

[0348] Each gRNA was validated in the mouse cell line, Neuro-N2a, by liposomal transient co-transfection with hSpCas9. 72 hours post-transfection genomic DNA was purified using QuickExtract DNA from Epicentre. PCR was performed to amplify the locus of interest. Subsequently the SURVEYOR Mutation Detection Kit from Transgenomics was followed. The SURVEYOR results for each gRNA and respective controls are shown in Figure A1. A positive SURVEYOR result is one large band corresponding to the genomic PCR and two smaller bands that are the product of the SURVEYOR nuclease making a double-strand break at the site of a mutation. The average cutting efficiency of each gRNA was also determined for each gRNA. The gRNA that was chosen for injection was the highest efficiency gRNA that was the most unique within the genome.

[0349] RNA (hSpCas9+gRNA RNA) was injected into the pronucleus of a zygote and later transplanted into a foster mother. Mothers were allowed to go full term and pups were sampled by tail snip 10 days postnatal. DNA was extracted and used as a template for PCR, which was then processed by SURVEYOR. Additionally, PCR products were sent for sequencing. Animals that were detected as being positive in either the SURVEYOR assay or PCR sequencing would have their genomic PCR products cloned into a pUC19 vector and sequenced to determine putative mutations from each allele.

[0350] So far, mice pups from the Chd8 targeting experiment have been fully processed up to the point of allele sequencing. The Surveyor results for 38 live pups (lanes 1-38) 1 dead pup (lane 39) and 1 wild-type pup for comparison (lane 40) are shown in Figure A2. Pups 1-19 were injected with gRNA Chd8.2 and pups 20-38 were injected with gRNA Chd8.3. Of the 38 live pups, 13 were positive for a mutation. The one dead pup also had a mutation. There was no mutation detected in the wild-type sample. Genomic PCR sequencing was consistent with the SURVEYOR assay findings.

Example 13

CRISPR/Cas-Mediated Transcriptional Modulation

[0351] FIG. 67 depicts a design of the CRISPR-TF (Transcription Factor) with transcriptional activation activity. The chimeric RNA is expressed by U6 promoter, while a human-codon-optimized, double-mutant version of the Cas9 protein (hSpCas9m), operably linked to triple NLS and a VP64 functional domain is expressed by a EF1.alpha. promoter. The double mutations, D10A and H840A, renders the cas9 protein unable to introduce any cleavage but maintained its capacity to bind to target DNA when guided by the chimeric RNA.

[0352] FIG. 68 depicts transcriptional activation of the human SOX2 gene with CRISPR-TF system (Chimeric RNA and the Cas9-NLS-VP64 fusion protein). 293FT cells were transfected with plasmids bearing two components: (1) U6-driven different chimeric RNAs targeting 20-bp sequences within or around the human SOX2 genomic locus, and (2) EF1a-driven hSpCas9m (double mutant)-NLS-VP64 fusion protein. 96 hours post transfection, 293FT cells were harvested and the level of activation is measured by the induction of mRNA expression using a qRT-PCR assay. All expression levels are normalized against the control group (grey bar), which represents results from cells transfected with the CRISPR-TF backbone plasmid without chimeric RNA. The qRT-PCR probes used for detecting the SOX2 mRNA is Taqman Human Gene Expression Assay (Life Technologies). All experiments represents data from 3 biological replicates, n=3, error bars show s.e.m.

Example 14

NLS: Cas9 NLS

[0353] 293FT cells were transfected with plasmid containing two components: (1) EF1.alpha. promoter driving the expression of Cas9 (wild-type human-codon-optimized Sp Cas9) with different NLS designs (2) U6 promoter driving the same chimeric RNA targeting human EMX1 locus.

[0354] Cells were collect at 72 h time point post transfection, and then extracted with 50 .mu.l of the QuickExtract genomic DNA extraction solution following manufacturer's protocol. Target EMX1 genomic DNA were PCR amplified and then Gel-purify with 1% agarose gel. Genomic PCR product were re-anneal and subjected to the Surveyor assay following manufacturer's protocol. The genomic cleavage efficiency of different constructs were measured using SDS-PAGE on a 4-12%0 TBE-PAGE gel (Life Technologies), analyzed and quantified with ImageLab (Bio-rad) software, all following manufacturer's protocol.

[0355] FIG. 69 depicts a design of different Cas9 NLS constructs. All Cas9 were the human-codon-optimized version of the Sp Cas9. NLS sequences are linked to the cas9 gene at either N-terminus or C-terminus. All Cas9 variants with different NLS designs were cloned into a backbone vector containing so it is driven by EF1a promoter. On the same vector there is a chimeric RNA targeting human EMX1 locus driven by U6 promoter, together forming a two-component system.

TABLE-US-00021 TABLE M Cas9 NLS Design Test Results. Quantification of genomic cleavage of different cas9-nls constructs by surveyor assay. Percentage Error Genome Biological Biological Biological (S.E.M., Cleavage as Replicate Replicate Replicate standard measured by 1 2 3 Average error of Surveyor assay (%) (%) (%) (%) the mean) Cas9 (No NLS) 2.50 3.30 2.73 2.84 0.24 Cas9 with N- term 7.61 6.29 5.46 6.45 0.63 NLS Cas9 with C-term 5.75 4.86 4.70 5.10 0.33 NLS Cas9 with Double 9.08 9.85 7.78 8.90 0.60 (N-term and C-term) NLS

[0356] FIG. 70 depicts the efficiency of genomic cleavage induced by Cas9 variants bearing different NLS designs. The percentage indicate the portion of human EMX1 genomic DNA that were cleaved by each construct. All experiments are from 3 biological replicates. n=3, error indicates S.E.M.

Example 15

Engineering of a Microalgae Using Cas9

[0357] Methods of Delivering Cas9

[0358] Method 1: Applicants deliver Cas9 and guide RNA using a vector that expresses Cas9 under the control of a constitutive promoter such as Hsp70A-Rbc S2 or Beta2-tubulin.

[0359] Method 2: Applicants deliver Cas9 and T7 polymerase using vectors that expresses Cas9 and T7 polymerase under the control of a constitutive promoter such as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA will be delivered using a vector containing T7 promoter driving the guide RNA.

[0360] Method 3: Applicants deliver Cas9 mRNA and in vitro transcribed guide RNA to algae cells. RNA can be in vitro transcribed. Cas9 mRNA will consist of the coding region for Cas9 as well as 3'UTR from Cop1 to ensure stabilization of the Cas9 mRNA.

[0361] For Homologous recombination, Applicants provide an additional homology directed repair template,

[0362] Sequence for a cassette driving the expression of Cas9 under the control of beta-2 tubulin promoter, followed by the 3' UTR. of Cop1.

TABLE-US-00022 (SEQ ID NO: 275) TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTG CGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGA CCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCT CCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGA CATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACT ACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTA AGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACAT GTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCG CAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACAT CGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAA GGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCA CAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGG CGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAG ATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTT CAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACT GGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCA CCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA GTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCAC CGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACAT GATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCC CGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGAC CTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGT GGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACG GCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGG CCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAA CTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCT GAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCA GATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCT GTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGA GATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGA CGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCA GCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAA GAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGA GTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCAC CGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAA GCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCT GGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCC ATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTT CCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAG ATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTG GAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTT CATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAA GGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTA TAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAA GCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCT GCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGA GGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTC CGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGA TCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGA AAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTT TGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCA CCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATA CACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCG GGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGA CGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAG CCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCA GGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCC CGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGA GCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGAT CGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAA CAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCT GGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCT GCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGA TATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTA CGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTC CATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAA GAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAA CTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAA GTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACT GGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCA GATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACAC TAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGAT CACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCA GTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGA CGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTA CCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTA CGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAA GGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTT CAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCC TCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAA GGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCA AGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAG CAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGC CAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAG CCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAA GGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGAT CACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTT TCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCAT CAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAA GAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACT GGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCA CTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACA GCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGA GCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAA TCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCC CATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGAC CAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCAT CGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCAC CCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGA CCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGT GGAGGCCAGCTAAGGATCCGGCAAGACTGGCCCCGCTTGGCAACG CAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTA TTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGC CTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACT

[0363] Sequence for a cassette driving the expression of 17 polymerase under the control of beta-2 tubulin promoter, followed by the 3' UTR of Cop1:

TABLE-US-00023 (SEQ ID NO: 276) TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTG CGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGA CCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCT CCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGA CATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACT ACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTA AGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACat gcctaagaagaagaggaaggttaacacgattaacatcgctaagaa cgacttctctgacatcgaactggctgctatcccgttcaacactct ggctgaccattacggtgagcgtttagctcgcgaacagttggccct tgagcatgagtcttacgagatgggtgaagcacgcttccgcaagat gtttgagcgtcaacttaaagctggtgaggttgcggataacgctgc cgccaagcctctcatcactaccctactccctaagatgattgcacg catcaacgactggtttgaggaagtgaaagctaagcgcggcaagcg cccgacagccttccagttcctgcaagaaatcaagccggaagccgt agcgtacatcaccattaagaccactctggcttgcctaaccagtgc tgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggc cattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagc taagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgt agggcacgtctacaagaaagcatttatgcaagttgtcgaggctga catgctctctaagggtctactcggtggcgaggcgtggtcttcgtg gcataaggaagactctattcatgtaggagtacgctgcatcgagat gctcattgagtcaaccggaatggttagcttacaccgccaaaatgc tggcgtagtaggtcaagactctgagactatcgaactcgcacctga atacgctgaggctatcgcaacccgtgcaggtgcgctggctggcat ctctccgatgttccaaccttgcgtagttcctcctaagccgtggac tggcattactggtggtggctattgggctaacggtcgtcgtcctct ggcgctggtgcgtactcacagtaagaaagcactgatgcgctacga agacgtttacatgcctgaggtgtacaaagcgattaacattgcgca aaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaa cgtaatcaccaagtggaagcattgtccggtcgaggacatccctgc gattgagcgtgaagaactcccgatgaaaccggaagacatcgacat gaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgt gtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttga gttcatgcttgagcaagccaataagtttgctaaccataaggccat ctggttcccttacaacatggactggcgcggtcgtgtttacgctgt gtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgct tacgctggcgaaaggtaaaccaatcggtaaggaaggttactactg gctgaaaatccacggtgcaaactgtgcgggtgtcgacaaggttcc gttccctgagcgcatcaagttcattgaggaaaaccacgagaacat catggcttgcgctaagtctccactggagaacacttggtgggctga gcaagattctccgttctgcttccttgcgttctgctttgagtacgc tggggtacagcaccacggcctgagctataactgctcccttccgct ggcgtttgacgggtcttgctctggcatccagcacttctccgcgat gctccgagatgaggtaggtggtcgcgcggttaacttgcttcctag tgaaaccgttcaggacatctacgggattgttgctaagaaagtcaa cgagattctacaagcagacgcaatcaatgggaccgataacgaagt agttaccgtgaccgatgagaacactggtgaaatctctgagaaagt caagctgggcactaaggcactggctggtcaatggctggcttacgg tgttactcgcagtgtgactaagcgttcagtcatgacgctggctta cgggtccaaagagttcggcttccgtcaacaagtgctggaagatac cattcagccagctattgattccggcaagggtctgatgttcactca gccgaatcaggctgctggatacatggctaagctgatttgggaatc tgtgagcgtgacggtggtagctgcggttgaagcaatgaactggct taagtctgctgctaagctgctggctgctgaggtcaaagataagaa gactggagagattcttcgcaagcgttgcgctgtgcattgggtaac tcctgatggtttccctgtgtggcaggaatacaagaagcctattca gacgcgcttgaacctgatgttcctcggtcagttccgcttacagcc taccattaacaccaacaaagatagcgagattgatgcacacaaaca ggagtctggtatcgctcctaacttgtacacagccaagacggtagc caccttcgtaagactgtagtgtgggcacacgagaagtacggaatc gaatcttttgcactgattcacgactccttcggtacgattccggct gacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgac acatatgagtcttgtgatgtactggctgatttctacgaccagttc gctgaccagttgcacgagtctcaattggacaaaatgccagcactt ccggctaaaggtaacttgaacctccgtgacatcttagagtcggac ttcgcgttcgcgtaaGGATCCGGCAAGACTGGCCCCGCTTGGCAA CGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATG TATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCC GCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACT

[0364] Sequence of guide RNA driven In 7 promoter (T7 promoter, Ns represent targeting sequence):

TABLE-US-00024 (SEQ ID NO: 277) gaaatTAATACGACTCACTATANNNNNNNNNNNNNNNNNNNNgtt ttagagctaGAAAtagcaagttaaaataaggctagtccgttatca acttgaaaaagtggcaccgagtcggtgcttttttt

[0365] Gene Delivery:

[0366] Chlamydomonas reinhardtii strain CC-124 and CC-125 from the Chlamydomonas Resource Center will be used for electroporation. Electroporation protocol follows standard recommended protocol from the GeneArt Chlamydomonas Engineering kit.

[0367] Also, Applicants generate a line of Chlamydomonas reinhardtii that expresses Cas9 constitutively. This can be done by using pChlamyl (linearized using PvuI) and selecting for hygromycin resistant colonies. Sequence for pChlamyl containing Cas9 is below. In this way to achieve gene knockout one simply needs to deliver RNA for the guideRNA. For homologous recombination Applicants deliver guideRNA as well as a linearized homologous recombination template.

TABLE-US-00025 pChlamy1-Cas9: (SEQ ID NO: 278) TGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGT GCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATAT GTATCCGCTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCT TTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGA GTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACT CCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGG CCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCC AGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAG AAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTG TTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCG CAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTC GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT CGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATC ACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCC ATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGAT CTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTG AGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGC GACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTA TTGAAGCATTTATCAGGGTTATTGTCTCATGACCAAAATCCCTTA ACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAT CAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCC GGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG CAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTT AGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGC TCTGCTAATCCTGTTACCAGTGGCTGTTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGC GCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTT GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTA TCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCT TCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTT CCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTT ATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGC TGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT GAGCGAGGAAGCGGTCGCTGAGGCTTGACATGATTGGTGCGTATG TTTGTATGAAGCTACAGGACTGATTTGGCGGGCTATGAGGGCGGG GGAAGCTCTGGAAGGGCCGCGATGGGGCGCGCGGCGTCCAGAAGG CGCCATACGGCCCGCTGGCGGCACCCATCCGGTATAAAAGCCCGC GACCCCGAACGGTGACCTCCACTTTCAGCGACAAACGAGCACTTA TACATACGCGACTATTCTGCCGCTATACATAACCACTCAGCTAGC TTAAGATCCCATCAAGCTTGCATGCCGGGCGCGCCAGAAGGAGCG CAGCCAAACCAGGATGATGTTTGATGGGGTATTTGAGCACTTGCA ACCCTTATCCGGAAGCCCCCTGGCCCACAAAGGCTAGGCGCCAAT GCAAGCAGTTCGCATGCAGCCCCTGGAGCGGTGCCCTCCTGATAA ACCGGCCAGGGGGCCTATGTTCTTTACTTTTTTACAAGAGAAGTC ACTCAACATCTTAAAATGGCCAGGTGAGTCGACGAGCAAGCCCGG CGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCAT TGTGTCGACGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAG CATCTAACCCTGCGTCGCCGTTTCCATTTGCAGGAGATTCGAGGT ACCATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAA AAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTG GACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAG TACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGAC CGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC AGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGA AGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAG ATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCAC AGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAG CGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCAC GAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGAC AGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCC CACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTG AACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTG CAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGC GGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGC AGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAG AATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACC CCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTG CAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTG GCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAG AACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAAC ACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGA TACGACGAGCACCACCAGGACCTGACCCTGCTGAAAATCTGATCG CCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGA TTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCG ACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACG ACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACG CCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGC TGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCC TGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACC TGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGT ACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCT ACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCA AGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGA AGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACA ACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCA TTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACC GGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACG TGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCA GAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGG TGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCA ACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACA GCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAG TGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCG GCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACC GGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAA TCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGT TCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCA AGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGG AAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGA TCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAG TGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGC TGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCA AGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAA ACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGG ACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACG AGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCA TCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGG GCCGGCACAACTCCCGAGAACATCGTGATCGAAATGGCCAGAGAG AACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATG AAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTG AAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTG TACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAG GAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATC GTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTG

CTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCC TCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTG CTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACC AAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTC ATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTG GCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAAT GACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAG CTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGC GAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCC GTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGC GAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATG ATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTAC TTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACC CTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAAC GGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCC ACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAA AAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTG CCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGG GACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTAT TCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAA CTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGA AGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGC TACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTAC TCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCT GCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAA TATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAG GGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAG CACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTC TCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTG TCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCC GAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCT GCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTAC ACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGC ATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGA GGCGACAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCTAACAT ATGATTCGAATGTCTTTCTTGCGCTATGACACTTCCAGCAAAAGG TAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCG ATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGC TCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCC CGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAAC ACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGAT CGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACA ACCCGCAAACATGACACAAGAATCCCTGTTACTTCTCGACCGTAT TGATTCGGATGATTCCTACGCGAGCCTGCGGAACGACCAGGAATT CTGGGAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTG CTTGCAGATTTGACTTGCAACGCCCGCATTGTGTCGACGAAGGCT TTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCCTGCGTC GCCGTTTCCATTTGCAGCCGCTGGCCCGCCGAGCCCTGGAGGAGC TCGGGCTGCCGGTGCCGCCGGTGCTGCGGGTGCCCGGCGAGAGCA CCAACCCCGTACTGGTCGGCGAGCCCGGCCCGGTGATCAAGCTGT TCGGCGAGCACTGGTGCGGTCCGGAGAGCCTCGCGTCGGAGTCGG AGGCGTACGCGGTCCTGGCGGACGCCCCGGTGCCGGTGCCCCGCC TCCTCGGCCGCGGCGAGCTGCGGCCCGGCACCGGAGCCTGGCCGT GGCCCTACCTGGTGATGAGCCGGATGACCGGCACCACCTGGCGGT CCGCGATGGACGGCACGACCGACCGGAACGCGCTGCTCGCCCTGG CCCGCGAACTCGGCCGGGTGCTCGGCCGGCTGCACAGGGTGCCGC TGACCGGGAACACCGTGCTCACCCCCCATTCCGAGGTCTTCCCGG AACTGCTGCGGGAACGCCGCGCGGCGACCGTCGAGGACCACCGCG GGTGGGGCTACCTCTCGCCCCGGCTGCTGGACCGCCTGGAGGACT GGCTGCCGGACGTGGACACGCTGCTGGCCGGCCGCGAACCCCGGT TCGTCCACGGCGACCTGCACGGGACCAACATCTTCGTGGACCTGG CCGCGACCGAGGTCACCGGGATCGTCGACTTCACCGACGTCTATG CGGGAGACTCCCGCTACAGCCTGGTGCAACTGCATCTCAACGCCT TCCGGGGCGACCGCGAGATCCTGGCCGCGCTGCTCGACGGGGCGC AGTGGAAGCGGACCGAGGACTTCGCCCGCGAACTGCTCGCCTTCA CCTTCCTGCACGACTTCGAGGTGTTCGAGGAGACCCCGCTGGATC TCTCCGGCTTCACCGATCCGGAGGAACTGGCGCAGTTCCTCTGGG GGCCGCCGGACACCGCCCCCGGCGCCTGATAAGGATCCGGCAAGA CTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGT TTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACC CGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAAC GTCAGTTGATGGTACT

[0368] For all modified Chlatnydomonas reinhardtii cells, Applicants used PCR, SURVEYOR nuclease assay, and DNA sequencing to verify successful modification.

Example 16

Use of Cas9 as a Transcriptional Repressor in Bacteria

[0369] The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Applicants describe here the use of the RNA-guided Cas9 protein as a programmable transcriptional repressor.

[0370] Applicants have previously demonstrated how the Cas9 protein of Streptococcus pyogenes SF370 can be used to direct genome editing in Streptococcus pneumoniae. In this study Applicants engineered the crR6Rk strain containing a minimal CRISPR system, consisting of cas9, the tracrRNA and a repeat. The DIOA-H840 mutations were introduced into cas9 in this strain, giving strain crR6Rk**. Four spacers targeting different positions of the bga4 P3-galactosidase gene promoter were cloned in the CRISPR array carried by the previously described pDB98 plasmid. Applicants observed a X to Y fold reduction in j3-galactosidase activity depending on the targeted position, demonstrating the potential of Cas9 as a programmable repressor (FIG. 73).

[0371] To achieve Cas9** repression in Escherichia coli a green fluorescence protein (GFP) reporter plasimd (pDB127) was constructred to express the gfpmu12 gene from a constituitive promoter. The promoter was designed to carry several NPP PAMs on both strands, to measure the effect of Cas9** binding at various positions. Applicants introduced the D10A-H840 mutations into pCas9, a plasmid described carrying the tracrRNA, cas9 and a minimal CRISPR array designed for the easy cloning of new spacers. Twenty-two different spacers were designed to target different regions of the gfpmut2 promoter and open reading frame. An approximately 20-fold reduction of fluorescence of was observed upon targeting regions overlapping or adjacent to the -35 and -10 promoter elements and to the Shine-Dalgarno sequence. Targets on both strands showed similar repression levels. These results suggest that the binding of Cas9** to any position of the promoter region prevents transcription initiation, presumably through steric inhibition of RNAP binding.

[0372] To determine whether Cas9** could prevent transcription elongation, Applicants directed it to the reading frame of gpfmut2. A reduction in fluorescence was observed both when the coding and non-coding strands where targeted, suggesting that Cas9 binding is actually strong enough to represent an obstacle to the running RNAP. However, while a 40% reduction in expression was observed when the coding strand was the target, a 20-fold reduction was observed for the non-coding strand (FIG. 21b, compare T9, T10 and T11 to B9, B10 and B11). To directly determine the effects of Cas9** binding on transcription, Applicants extracted RNA from strains carrying either the T5, T10, B10 or a control construct that does not target pDB127 and subjected it to Northern blot analysis using either a probe binding before (B477) or after (B510) the B10 and T10 target sites. Consistent with Applicants' fluorescence methods, no g4)mut2 transcription was detected when Cas9** was directed to the promoter region (T5 target) and a transcription was observed after the targeting of the T10 region. Interestingly, a smaller transcript was observed with the B477 probe. This band corresponds to the expected size of a transcript that would be interrupted by Cas9**, and is a direct indication of a transcriptional termination caused by dgRNA::Cas9** binding to the coding strand. Surprisingly, Applicants detected no transcript when the non-coding strand was targeted (B10). Since Cas9** binding to the B10 region is unlikely to interfere with transcription initiation, this result suggests that the mRNA was degraded. DgRNA::Cas9 was shown to bind ssRNA in vitro. Applicants speculate that binding may trigger degradation of the mRNA by host nucleases. Indeed, ribosome stalling can induce cleavage on the translated mRNA in E. coli.

[0373] Some applications require a precise tuning gene expression rather than its complete repression. Applicants sought to achieve intermediate repression levels through the introduction of mismatches that will weaken the crRNA/target interactions. Applicants created a series of spacers based on the B1, T5 and B10 constructs with increasing numbers of mutations in the 5' end of the crRNA. Up to 8 mutations in B1 and T5 did not affect the repression level, and a progressive increased in fluorescence was observed for additional mutations.

[0374] The observed repression with only an 8 nt match between the crRNA and its target raises the question of off-targeting effects of the use of Cas9** as a transcriptional regulator. Since a good PAM (NGG) is also required for Cas9 binding, the number of nucleotides to match to obtain some level of respiration is 10. A 10 nt match occurs randomly once every .about.1 Mbp, and such sites are thus likely to be found even in small bacterial genomes. However, to effectively repress transcription, such site needs to be in the promoter region of gene, which makes off-targeting much less likely. Applicants also showed that gene expression can be affected if the non-coding strand of a gene is targeted. For this to happen, a random target would have to be in the right orientation, but such events relatively more likely to happen. As a matter of fact, during the course of this study Applicants were unable to construct one of the designed spacer on pCas9**. Applicants later found this spacer showed a 12 bp match next to a good PAM in the essential murC gene. Such off-targeting could easily be avoided by a systematic blast of the designed spacers.

[0375] Aspects of the invention are further described in the following numbered paragraphs:

[0376] 1. A vector system comprising one or more vectors, wherein the system comprises

[0377] a. a first regulatory element operably linked to a traer mate sequence and one or more insertion sites for inserting a guide sequence upstream of the traer 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 traer mate sequence that is hybridized to the traer sequence; and

[0378] b. a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence;

[0379] wherein components (a) and (b) are located on the same or different vectors of the system.

[0380] 2. The vector system of paragraph 1, wherein component (a) further comprises the traer sequence downstream of the traer mate sequence under the control of the first regulatory element.

[0381] 3. The vector system of paragraph 1, wherein 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.

[0382] 4. The vector system of paragraph 1, wherein the system comprises the traer sequence under the control of a third regulatory element.

[0383] 5. The vector system of paragraph 1, wherein the traer sequence exhibits at least 50% of sequence complementarity along the length of the traer mate sequence when optimally aligned.

[0384] 6. The vector system of paragraph 1, wherein 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.

[0385] 7. The vector system of paragraph 1, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

[0386] 8. The vector system of paragraph 1, wherein the CRISPR enzyme is a Cas9 enzyme.

[0387] 9. The vector system of paragraph 1, wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.

[0388] 10. The vector system of paragraph 1, wherein the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence.

[0389] 11. The vector system of paragraph 1, wherein the CRISPR enzyme lacks DNA strand cleavage activity.

[0390] 12. The vector system of paragraph 1, wherein the first regulatory element is a polymerase III promoter.

[0391] 13. The vector system of paragraph 1, wherein the second regulatory element is a polymerase II promoter.

[0392] 14. The vector system of paragraph 4, wherein the third regulatory element is a polymerase III promoter.

[0393] 15. The vector system of paragraph 1, wherein the guide sequence is at least 15 nucleotides in length.

[0394] 16. The vector system of paragraph 1, wherein fewer than 50% of the nucleotides of the guide sequence participate in self-complementary base-pairing when optimally folded.

[0395] 17. A vector comprising a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising one or more nuclear localization sequences, wherein 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.

[0396] 18. The vector of paragraph 17, wherein said regulatory element is a polymerase II promoter.

[0397] 19. The vector of paragraph 17, wherein said CRISPR enzyme is a type IICRISPR system enzyme.

[0398] 20. The vector of paragraph 17, wherein said CRISPR enzyme is a Cas9 enzyme.

[0399] 21. The vector of paragraph 17, wherein said CRISPR enzyme lacks the ability to cleave one or more strands of a target sequence to which it binds.

[0400] 22. 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.

[0401] 23. The CRISPR enzyme of paragraph 22, wherein said CRISPR enzyme is a type IICRISPR system enzyme.

[0402] 24. The CRISPR enzyme of paragraph 22, wherein said CRISPR enzyme is a Cas9 enzyme.

[0403] 25. The CRISPR enzyme of paragraph 22, wherein said CRISPR enzyme lacks the ability to cleave one or more strands of a target sequence to which it binds.

[0404] 26. A eukaryotic host cell comprising:

[0405] a. a first regulatory element operably linked to a traer mate sequence and one or more insertion sites for inserting a guide sequence upstream of the traer 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 traer mate sequence that is hybridized to the traer sequence; and/or

[0406] b. a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence.

[0407] 27. The eukaryotic host cell of paragraph 26, wherein said host cell comprises components (a) and (b).

[0408] 28. The eukaryotic host cell of paragraph 26, wherein component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell.

[0409] 29. The eukaryotic host cell of paragraph 26, wherein component (a) further comprises the traer sequence downstream of the traer mate sequence under the control of the first regulatory element.

[0410] 30. The eukaryotic host cell of paragraph 26, wherein 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.

[0411] 31. The eukaryotic host cell of paragraph 26, further comprising a third regulatory element operably linked to said traer sequence.

[0412] 32. The eukaryotic host cell of paragraph 26, wherein the traer sequence exhibits at least 50% of sequence complementarity along the length of the traer mate sequence when optimally aligned.

[0413] 33. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable mount in the nucleus of a eukaryotic cell.

[0414] 34. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

[0415] 35. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzyme is a Cas9 enzyme.

[0416] 36. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.

[0417] 37. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence.

[0418] 38. The eukaryotic host cell of paragraph 26, wherein the CRISPR enzyme lacks DNA strand cleavage activity.

[0419] 39. The eukaryotic host cell of paragraph 26, wherein the first regulatory element is a polymerase III promoter.

[0420] 40. The eukaryotic host cell of paragraph 26, wherein the second regulatory element is a polymerase II promoter.

[0421] 41. The eukaryotic host cell of paragraph 31, wherein the third regulatory element is a polymerase III promoter.

[0422] 42. The eukaryotic host cell of paragraph 26, wherein the guide sequence is at least 15 nucleotides in length.

[0423] 43. The eukaryotic host cell of paragraph 26, wherein fewer than 50% of the nucleotides of the guide sequence participate in self-complementary base-pairing when optimally folded.

[0424] 44. A non-human animal comprising a eukaryotic host cell of any one of paragraphs 26-43.

[0425] 45. A kit comprising a vector system and instructions for using said kit, the vector system comprising:

[0426] a. a first regulatory element operably linked to a traer mate sequence and one or more insertion sites for inserting a guide sequence upstream of the traer 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 traer mate sequence that is hybridized to the traer sequence; and/or

[0427] b. a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence.

[0428] 46. The kit of paragraph 45, wherein said kit comprises components (a) and (b) located on the same or different vectors of the system.

[0429] 47. The kit of paragraph 45, wherein component (a) further comprises the traer sequence downstream of the traer mate sequence under the control of the first regulatory element.

[0430] 48. The kit of paragraph 45, wherein 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.

[0431] 49. The kit of paragraph 45, wherein the system comprises the traer sequence under the control of a third regulatory element.

[0432] 50. The kit of paragraph 45, wherein the traer sequence exhibits at least 50% of sequence complementarity along the length of the traer mate sequence when optimally aligned.

[0433] 51. The kit of paragraph 45, wherein the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable mount in the nucleus of a eukaryotic cell.

[0434] 52. The kit of paragraph 45, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

[0435] 53. The kit of paragraph 45, wherein the CRISPR enzyme is a Cas9 enzyme.

[0436] 54. The kit of paragraph 45, wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.

[0437] 55. The kit of paragraph 45, wherein the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence.

[0438] 56. The kit of paragraph 45, wherein the CRISPR enzyme lacks DNA strand cleavage activity.

[0439] 57. The kit of paragraph 45, wherein the first regulatory element is a polymerase III promoter.

[0440] 58. The kit of paragraph 45, wherein the second regulatory element is a polymerase II promoter.

[0441] 59. The kit of paragraph 49, wherein the third regulatory element is a polymerase III promoter.

[0442] 60. The kit of paragraph 45, wherein the guide sequence is at least 15 nucleotides in length.

[0443] 61. The kit of paragraph 45, wherein fewer than 50% of the nucleotides of the guide sequence participate in self-complementary base-pairing when optimally folded.

[0444] 62. A computer system for selecting a candidate target sequence within a nucleic acid sequence in a eukaryotic cell for targeting by a CRISPR complex, the system comprising:

[0445] a. a memory unit configured to receive and/or store said nucleic acid sequence; and

[0446] b. one or more processors alone or in combination programmed to (i) locate a CRISPR motif sequence within said nucleic acid sequence, and (ii) select a sequence adjacent to said located CRISPR motif sequence as the candidate target sequence to which the CRISPR complex binds.

[0447] 63. The computer system of paragraph 62, wherein said locating step comprises identifying a CRISPR motif sequence located less than about 500 nucleotides away from said target sequence.

[0448] 64. The computer system of paragraph 62, wherein said candidate target sequence is at least 10 nucleotides in length.

[0449] 65. The computer system of paragraph 62, wherein the nucleotide at the 3' end of the candidate target sequence is located no more than about 10 nucleotides upstream of the CRISPR motif sequence.

[0450] 66. The computer system of paragraph 62, wherein the nucleic acid sequence in the eukaryotic cell is endogenous to the eukaryotic genome.

[0451] 67. The computer system of claim 62, wherein the nucleic acid sequence in the eukaryotic cell is exogenous to the eukaryotic genome.

[0452] 68. A computer-readable medium comprising codes that, upon execution by one or more processors, implements a method of selecting a candidate target sequence within a nucleic acid sequence in a eukaryotic cell for targeting by a CRISPR complex, said method comprising: (a) locating a CRISPR motif sequence within said nucleic acid sequence, and (b) selecting a sequence adjacent to said located CRISPR motif sequence as the candidate target sequence to which the CRISPR complex binds.

[0453] 69. The computer-readable medium of paragraph 68, wherein said locating comprises locating a CRISPR motif sequence that is less than about 500 nucleotides away from said target sequence.

[0454] 70. The computer-readable of paragraph 68, wherein said candidate target sequence is at least 10 nucleotides in length.

[0455] 71. The computer-readable of paragraph 68, wherein the nucleotide at the 3' end of the candidate target sequence is located no more than about 10 nucleotides upstream of the CRISPR motif sequence.

[0456] 72. The computer-readable of paragraph 68, wherein the nucleic acid sequence in the eukaryotic cell is endogenous the eukaryotic genome.

[0457] 73. The computer-readable of paragraph 68, wherein the nucleic acid sequence in the eukaryotic cell is exogenous to the eukaryotic genome.

[0458] 74. A method of modifying a target polynucleotide in a eukaryotic cell, the method comprising 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 traer mate sequence which in tum hybridizes to a traer sequence.

[0459] 75. The method of paragraph 74, wherein said cleavage comprises cleaving one or two strands at the location of the target sequence by said CRISPR enzyme.

[0460] 76. The method of paragraph 74, wherein said cleavage results in decreased transcription of a target gene.

[0461] 77. The method of paragraph 74, further comprising 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.

[0462] 78. The method of paragraph 77, wherein said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.

[0463] 79. The method of paragraph 74, further comprising 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 traer mate sequence, and the traer sequence.

[0464] 80. The method of paragraph 79, wherein said vectors are delivered to the eukaryotic cell in a subject.

[0465] 81. The method of paragraph 74, wherein said modifying takes place in said eukaryotic cell in a cell culture.

[0466] 82. The method of paragraph 74, further comprising isolating said eukaryotic cell from a subject prior to said modifying.

[0467] 83. The method of paragraph 82, further comprising returning said eukaryotic cell and/or cells derived therefrom to said subject.

[0468] 84. A method of modifying expression of a polynucleotide in a eukaryotic cell, the method comprising: 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 traer mate sequence which in tum hybridizes to a traer sequence.

[0469] 85. The method of paragraph 74, further comprising 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 traer mate sequence, and the traer sequence.

[0470] 86. A method of generating a model eukaryotic cell comprising a mutated disease gene, the method comprising:

[0471] 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 traer mate sequence, and a traer sequence; and

[0472] 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 traer mate sequence that is hybridized to the traer sequence, thereby generating a model eukaryotic cell comprising a mutated disease gene.

[0473] 87. The method of paragraph 86, wherein said cleavage comprises cleaving one or two strands at the location of the target sequence by said CRISPR enzyme.

[0474] 88. The method of paragraph 86, wherein said cleavage results in decreased transcription of a target gene.

[0475] 89. The method of paragraph 86, further comprising 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.

[0476] 90. The method of paragraph 89, wherein said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.

[0477] 91. A method of developing a biologically active agent that modulates a cell signaling event associated with a disease gene, comprising:

[0478] a. contacting a test compound with a model cell of any one of paragraphs 86-90; and

[0479] 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.

[0480] 92. A recombinant polynucleotide comprising a guide sequence upstream of a traer 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.

[0481] 93. The recombinant polynucleotide of paragraph 89, wherein the target sequence is a viral sequence present in a eukaryotic cell.

[0482] 94. The recombinant polynucleotide of paragraph 89, wherein the target sequence is a proto-oncogene or an oncogene.

[0483] 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.

REFERENCES

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Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl. Acad. Sci. U.S.A. (2011). [0509] 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). [0510] 27. Zahner, D. & Hakenbeck, R. The Streptococcus pneumoniae beta-galactosidase is a surface protein. J. Bacteriol. 182, 5919-5921 (2000). [0511] 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). [0512] 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). [0513] 30. Hosaka, T. et al. 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Sequence CWU 1

1

53717PRTSimian virus 40 1Pro Lys Lys Lys Arg Lys Val 1 5 216PRTUnknownDescription of Unknown Nucleoplasmin bipartite NLS sequence 2Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 39PRTUnknownDescription of Unknown C-myc NLS sequence 3Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 411PRTUnknownDescription of Unknown C-myc NLS sequence 4Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro 1 5 10 538PRTHomo sapiens 5Asn 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 642PRTUnknownDescription of Unknown IBB domain from importin-alpha sequence 6Arg 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 78PRTUnknownDescription of Unknown Myoma T protein sequence 7Val Ser Arg Lys Arg Pro Arg Pro 1 5 88PRTUnknownDescription of Unknown Myoma T protein sequence 8Pro Pro Lys Lys Ala Arg Glu Asp 1 5 98PRTHomo sapiens 9Pro Gln Pro Lys Lys Lys Pro Leu 1 5 1012PRTMus musculus 10Ser Ala Leu Ile Lys Lys Lys Lys Lys Met Ala Pro 1 5 10 115PRTInfluenza virus 11Asp Arg Leu Arg Arg 1 5 127PRTInfluenza virus 12Pro Lys Gln Lys Lys Arg Lys 1 5 1310PRTHepatitus delta virus 13Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu 1 5 10 1410PRTMus musculus 14Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg 1 5 10 1520PRTHomo sapiens 15Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys 1 5 10 15 Lys Ser Lys Lys 20 1617PRTHomo sapiens 16Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys 1 5 10 15 Lys 1727DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 17nnnnnnnnnn nnnnnnnnnn nnagaaw 271819DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 18nnnnnnnnnn nnnnagaaw 191927DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 19nnnnnnnnnn nnnnnnnnnn nnagaaw 272018DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 20nnnnnnnnnn nnnagaaw 1821137DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 21nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcaagatt tagaaataaa tcttgcagaa 60gctacaaaga taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt 120tcgttattta atttttt 13722123DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 22nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcagaaat gcagaagcta caaagataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttcgt tatttaattt 120ttt 12323110DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 23nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcagaaat gcagaagcta caaagataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttttt 11024102DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 24nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc 60cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt 1022588DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 25nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc 60cgttatcaac ttgaaaaagt gttttttt 882676DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 26nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc 60cgttatcatt tttttt 762712DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 27gttttagagc ta 122831DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 28tagcaagtta aaataaggct agtccgtttt t 312927DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 29nnnnnnnnnn nnnnnnnnnn nnagaaw 273012RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 30guuuuagagc ua 123133DNAHomo sapiens 31ggacatcgat gtcacctcca atgactaggg tgg 333233DNAHomo sapiens 32cattggaggt gacatcgatg tcctccccat tgg 333333DNAHomo sapiens 33ggaagggcct gagtccgagc agaagaagaa ggg 333433DNAHomo sapiens 34ggtggcgaga ggggccgaga ttgggtgttc agg 333533DNAHomo sapiens 35atgcaggagg gtggcgagag gggccgagat tgg 333621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 36aaaaccaccc ttctctctgg c 213721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 37ggagattgga gacacggaga g 213820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 38ctggaaagcc aatgcctgac 203920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 39ggcagcaaac tccttgtcct 2040335DNAArtificial SequenceDescription 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 gaaccattca aaacagcata gcaagttaaa ataaggctag tccgttatca 300acttgaaaaa gtggcaccga gtcggtgctt ttttt 33541423DNAArtificial SequenceDescription 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 gtagtattaa gtattgtttt atggctgata aatttctttg aatttctcct 300tgattatttg ttataaaagt tataaaataa tcttgttgga accattcaaa acagcatagc 360aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 420ttt 42342339DNAArtificial SequenceDescription 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 ggttttagag ctatgctgtt ttgaatggtc ccaaaacggg tcttcgagaa 300gacgttttag agctatgctg ttttgaatgg tcccaaaac 33943309DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 43gagggcctat 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 309441648PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 44Met 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 451625PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 45Met 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 461664PRTArtificial SequenceDescription 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 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 471423PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 47Met 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 48483PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 48Met 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 49483PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 49Met 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 501423PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 50Met 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 512012DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 51gaatgctgcc 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 2012521153PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 52Met 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 53340DNAArtificial SequenceDescription 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 ttacttaaat cttgcagaag ctacaaagat aaggcttcat gccgaaatca 300acaccctgtc attttatggc agggtgtttt cgttatttaa 34054360DNAArtificial SequenceDescription 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 240cgaaacaccg ggttttagag ctatgctgtt ttgaatggtc ccaaaacnnn nnnnnnnnnn 300nnnnnnnnnn nnnnnnngtt ttagagctat gctgttttga atggtcccaa aacttttttt 36055318DNAArtificial SequenceDescription 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 gttttttt 31856325DNAArtificial SequenceDescription 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 gttatcattt ttttt 32557337DNAArtificial SequenceDescription 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 ttttttt 33758352DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 58gagggcctat 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 352595101DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 59cgttacataa 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 510160137DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 60nnnnnnnnnn nnnnnnnnnn gttattgtac tctcaagatt tagaaataaa tcttgcagaa 60gctacaaaga taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt 120tcgttattta atttttt 13761123DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 61nnnnnnnnnn nnnnnnnnnn gttattgtac tctcagaaat gcagaagcta caaagataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttcgt tatttaattt 120ttt 12362110DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 62nnnnnnnnnn nnnnnnnnnn gttattgtac tctcagaaat gcagaagcta caaagataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttttt 11063137DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 63nnnnnnnnnn nnnnnnnnnn gttattgtac tctcaagatt tagaaataaa tcttgcagaa 60gctacaatga taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt 120tcgttattta atttttt 13764123DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 64nnnnnnnnnn nnnnnnnnnn gttattgtac tctcagaaat gcagaagcta caatgataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttcgt tatttaattt 120ttt 12365110DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 65nnnnnnnnnn nnnnnnnnnn gttattgtac tctcagaaat gcagaagcta caatgataag 60gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttttt 11066107DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 66nnnnnnnnnn nnnnnnnnnn gttttagagc tgtggaaaca cagcgagtta aaataaggct 60tagtccgtac tcaacttgaa aaggtggcac cgattcggtg ttttttt 107674263DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 67atgaaaaggc 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 42636853DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 68tcctagcagg atttctgata ttactgtcac gttttagagc tatgctgttt tga 536953DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 69gtgacagtaa tatcagaaat cctgctagga gttttgggac cattcaaaac agc 537025DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 70gggtttcaag tctttgtagc aagag 257124DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 71gccaatgaac gggaaccctt ggtc 247225DNAArtificial SequenceDescription of

Artificial Sequence Synthetic primer 72nnnngacgag gcaatggctg aaatc 257325DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 73nnnnttattt ggctcatatt tgctg 257425DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 74ctttacacca atcgctgcaa cagac 257545DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 75caaaatttct agtcttcttt gcctttcccc ataaaaccct cctta 457645DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 76agggttttat ggggaaaggc aaagaagact agaaattttg atacc 457725DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 77cttacggtgc ataaagtcaa tttcc 257821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 78tggctcgatt tcagccattg c 217943DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 79ctttgacgag gcaatggctg aaatcgagcc aanaaagcgc aag 438043DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 80ctttgacgag gcaatggctg aaatcgagcc aaanaagcgc aag 438143DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 81ctttgacgag gcaatggctg aaatcgagcc aaaanagcgc aag 438243DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 82ctttgacgag gcaatggctg aaatcgagcc aaaaangcgc aag 438343DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 83ctttgacgag gcaatggctg aaatcgagcc aaaaaancgc aag 438443DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 84ctttgacgag gcaatggctg aaatcgagcc aaaaaagngc aag 438546DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 85ctttgacgag gcaatggctg aaatcgagcc aaaaaagcnc aagaag 468646DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 86ctttgacgag gcaatggctg aaatcgagcc aaaaaagcgn aagaag 468746DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 87ctttgacgag gcaatggctg aaatcgagcc aaaaaagcgc nagaag 468823DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 88gcgctttttt ggctcgattt cag 238940DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 89caatggctga aatcgagcca aaaaagcgca ngaagaaatc 409040DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 90caatggctga aatcgagcca aaaaagcgca anaagaaatc 409140DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 91caatggctga aatcgagcca aaaaagcgca agnagaaatc 409240DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 92caatggctga aatcgagcca aaaaagcgca agangaaatc 409340DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 93caatggctga aatcgagcca aaaaagcgca agaanaaatc 409444DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 94caatggctga aatcgagcca aaaaagcgca agaagnaatc aacc 449544DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 95caatggctga aatcgagcca aaaaagcgca agaaganatc aacc 449644DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 96caatggctga aatcgagcca aaaaagcgca agaagaantc aacc 449744DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 97caatggctga aatcgagcca aaaaagcgca agaagaaanc aacc 449847DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 98caatggctga aatcgagcca aaaaagcgca agaagaaatn aaccagc 479947DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 99caatggctga aatcgagcca aaaaagcgca agaagaaatc naccagc 4710031DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 100gatcctccat ccgtacaacc cacaaccctg g 3110131DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 101aattccaggg ttgtgggttg tacggatgga g 3110234DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 102catggatcct atttcttaat aactaaaaat atgg 3410333DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 103catgaattca actcaacaag tctcagtgtg ctg 3310435DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 104aaacattttt tctccattta ggaaaaagga tgctg 3510535DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 105aaaacagcat cctttttcct aaatggagaa aaaat 3510635DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 106aaaccttaaa tcagtcacaa atagcagcaa aattg 3510735DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 107aaaacaattt tgctgctatt tgtgactgat ttaag 3510835DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 108aaacttttca tcatacgacc aatctgcttt atttg 3510935DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 109aaaacaaata aagcagattg gtcgtatgat gaaaa 3511035DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 110aaactcgtcc agaagttatc gtaaaagaaa tcgag 3511135DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 111aaaactcgat ttcttttacg ataacttctg gacga 3511235DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 112aaacaatctc tccaaggttt ccttaaaaat ctctg 3511335DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 113aaaacagaga tttttaagga aaccttggag agatt 3511435DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 114aaacgccatc gtcaggaaga agctatgctt gagtg 3511535DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 115aaaacactca agcatagctt cttcctgacg atggc 3511635DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 116aaacatctct atacttattg aaatttcttt gtatg 3511735DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 117aaaacataca aagaaatttc aataagtata gagat 3511835DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 118aaactagctg tgatagtccg caaaaccagc cttcg 3511935DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 119aaaacgaagg ctggttttgc ggactatcac agcta 3512035DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 120aaacatcgga aggtcgagca agtaattatc ttttg 3512135DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 121aaaacaaaag ataattactt gctcgacctt ccgat 3512235DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 122aaacaagatg gtatcgcaaa gtaagtgaca ataag 3512335DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 123aaaacttatt gtcacttact ttgcgatacc atctt 3512452DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 124gagacctttg agcttccgag actggtctca gttttgggac cattcaaaac ag 5212552DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 125tgagaccagt ctcggaagct caaaggtctc gttttagagc tatgctgttt tg 5212635DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 126aaactacttt acgcagcgcg gagttcggtt ttttg 3512735DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 127aaaacaaaaa accgaactcc gcgctgcgta aagta 3512845DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 128atgccggtac tgccgggcct cttgcgggat tacgaaatca tcctg 4512945DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 129gtgactggcg atgctgtcgg aatggacgat cacactactc ttctt 4513050DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 130ttaagaaata atcttcatct aaaatatact tcagtcacct cctagctgac 5013148DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 131attgatttga gtcagctagg aggtgactga agtatatttt agatgaag 4813285DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 132gagacctttg agcttccgag actggtctca gttttgggac cattcaaaac agcatagctc 60taaaacctcg tagactattt ttgtc 8513384DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 133gagaccagtc tcggaagctc aaaggtctcg ttttagagct atgctgtttt gaatggtccc 60aaaacttcag cacactgaga cttg 8413421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 134agtcatccca gcaacaaatg g 2113531DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 135cgtggtaaat cggataacgt tccaagtgaa g 3113622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 136tgctcttctt cacaaacaag gg 2213721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 137aagccaaagt ttggcaccac c 2113822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 138gtagcttatt cagtcctagt gg 2213945DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 139cgtttgttga actaatgggt gcaaattacg aatcttctcc tgacg 4514045DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 140cgtcaggaga agattcgtaa tttgcaccca ttagttcaac aaacg 4514148DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 141gatattatgg agcctatttt tgtgggtttt taggcataaa actatatg 4814248DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 142catatagttt tatgcctaaa aacccacaaa aataggctcc ataatatc 4814327DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 143attatttctt aataactaaa aatatgg 2714424DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 144cgtgtacaat tgctagcgta cggc 2414524DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 145gcaccggtga tcactagtcc tagg 2414647DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 146cctaggacta gtgatcaccg gtgcaaatat gagccaaata aatatat 4714744DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 147gccgtacgct agcaattgta cacgtttgtt gaactaatgg gtgc 4414825DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 148ttcaaatttt cccatttgat tctcc 2514947DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 149ccatattttt agttattaag aaataatacc agccatcagt cacctcc 4715023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 150agacgattca atagacaata agg 2315145DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 151gttttgggac cattcaaaac agcatagctc taaaacctcg tagac 4515250DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 152gctatgctgt tttgaatggt cccaaaacca ttattttaac acacgaggtg 5015350DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 153gctatgctgt tttgaatggt cccaaaacgc acccattagt tcaacaaacg 5015423DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 154aattcttttc ttcatcatcg gtc 2315524DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 155aagaaagaat gaagattgtt catg 2415625DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 156ggtactaatc aaaatagtga ggagg 2515723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 157gtttttcaaa atctgcggtt gcg 2315826DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 158aaaaattgaa aaaatggtgg aaacac 2615953DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 159atttcgtaaa cggtatcggt ttcttttaaa gttttgggac cattcaaaac agc 5316053DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 160tttaaaagaa accgataccg tttacgaaat gttttagagc tatgctgttt tga 5316153DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 161aaacggtatc ggtttctttt aaattcaatt gttttgggac cattcaaaac agc 5316253DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 162aattgaattt aaaagaaacc gataccgttt gttttagagc tatgctgttt tga 5316339DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 163gttccttaaa ccaaaacggt atcggtttct tttaaattc 3916447DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 164gaaaccgata ccgttttggt ttaaggaaca ggtaaagggc atttaac 4716522DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 165cgatttcagc cattgcctcg tc 2216656DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 166gcctttgacg aggcaatggc tgaaatcgnn nnnaaaaagc gcaagaagaa atcaac 5616753DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 167tccgtacaac ccacaaccct gctagtgagc gttttgggac cattcaaaac agc 5316853DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 168gctcactagc agggttgtgg gttgtacgga gttttagagc tatgctgttt tga 5316923DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 169ttgttgccac tcttccttct ttc 2317041DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 170cagggttgtg ggttgttgcg atggagttaa ctcccatctc c 4117141DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 171gggagttaac tccatcgcaa caacccacaa ccctgctagt g 4117222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 172gtggtatcta tcgtgatgtg ac 2217323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 173ttaccgaaac ggaatttatc tgc 2317422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 174aaagctagag ttccgcaatt gg 2217537DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 175gtgggttgta cggattgagt taactcccat ctccttc 3717638DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 176gatgggagtt aactcaatcc gtacaaccca caaccctg 3817740DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 177gcttcaccta ttgcagcacc aattgaccac atgaagatag 4017841DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 178gtggtcaatt ggtgctgcaa taggtgaagc taatggtgat g 4117939DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 179ctgatttgta ttaattttga gacattatgc ttcaccttc 3918040DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 180gcataatgtc tcaaaattaa tacaaatcag tgaaatcatg 4018152DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 181gttttgggac cattcaaaac agcatagctc taaaacgtga cagtaatatc ag

5218253DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 182gttttagagc tatgctgttt tgaatggtcc caaaacgctc actagcaggg ttg 5318359DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 183atactttacg cagcgcggag ttcggttttg taggagtggt agtatataca cgagtacat 5918433DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 184gctcactagc agggttgtgg gttgtacgga tgg 3318533DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 185tcctagcagg atttctgata ttactgtcac tgg 3318633DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 186tttaaaagaa accgataccg tttacgaaat tgg 3318784DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 187ggaaccattc ataacagcat agcaagttat aataaggcta gtccgttatc aacttgaaaa 60agtggcaccg agtcggtgct tttt 8418836DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 188gttatagagc tatgctgtta tgaatggtcc caaaac 3618984DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 189ggaaccattc aatacagcat agcaagttaa tataaggcta gtccgttatc aacttgaaaa 60agtggcaccg agtcggtgct tttt 8419036DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 190gtattagagc tatgctgtat tgaatggtcc caaaac 36191103DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 191nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc 60cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttt 103192103DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 192nnnnnnnnnn nnnnnnnnnn gtattagagc tagaaatagc aagttaatat aaggctagtc 60cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttt 103193123DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 193nnnnnnnnnn nnnnnnnnnn gttttagagc tatgctgttt tggaaacaaa acagcatagc 60aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 120ttt 123194123DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 194nnnnnnnnnn nnnnnnnnnn gtattagagc tatgctgtat tggaaacaat acagcatagc 60aagttaatat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 120ttt 12319520DNAHomo sapiens 195gtcacctcca atgactaggg 2019623DNAHomo sapiens 196gacatcgatg tcctccccat tgg 2319723DNAHomo sapiens 197gagtccgagc agaagaagaa ggg 2319823DNAHomo sapiens 198gcgccaccgg ttgatgtgat ggg 2319923DNAHomo sapiens 199ggggcacaga tgagaaactc agg 2320023DNAHomo sapiens 200gtacaaacgg cagaagctgg agg 2320123DNAHomo sapiens 201ggcagaagct ggaggaggaa ggg 2320223DNAHomo sapiens 202ggagcccttc ttcttctgct cgg 2320323DNAHomo sapiens 203gggcaaccac aaacccacga ggg 2320423DNAHomo sapiens 204gctcccatca catcaaccgg tgg 2320523DNAHomo sapiens 205gtggcgcatt gccacgaagc agg 2320623DNAHomo sapiens 206ggcagagtgc tgcttgctgc tgg 2320723DNAHomo sapiens 207gcccctgcgt gggcccaagc tgg 2320823DNAHomo sapiens 208gagtggccag agtccagctt ggg 2320923DNAHomo sapiens 209ggcctcccca aagcctggcc agg 2321023DNAHomo sapiens 210ggggccgaga ttgggtgttc agg 2321123DNAHomo sapiens 211gtggcgagag gggccgagat tgg 2321223DNAHomo sapiens 212gagtgccgcc gaggcggggc ggg 2321323DNAHomo sapiens 213ggagtgccgc cgaggcgggg cgg 2321423DNAHomo sapiens 214ggagaggagt gccgccgagg cgg 2321520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 215ccatcccctt ctgtgaatgt 2021620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 216ggagattgga gacacggaga 2021720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 217aagcaccgac tcggtgccac 2021820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 218tcacctccaa tgactagggg 2021922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 219caagttgata acggactagc ct 2222023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 220agtccgagca gaagaagaag ttt 2322125DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 221tttcaagttg ataacggact agcct 2522220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 222aaacagcaga ttcgcctgga 2022320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 223tcatccgctc gatgaagctc 2022420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 224tccaaaatca agtggggcga 2022520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 225tgatgaccct tttggctccc 2022645DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 226gaggaattct ttttttgtty gaatatgttg gaggtttttt ggaag 4522742DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 227gagaagctta aataaaaaac racaatactc aacccaacaa cc 4222817DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 228caggaaacag ctatgac 1722939DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 229gcctctagag gtacctgagg gcctatttcc catgattcc 39230133DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 230acctctagaa aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct 60tattttaact tgctatttct agctctaaaa cnnnnnnnnn nnnnnnnnnn nggtgtttcg 120tcctttccac aag 133231133DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 231acctctagaa aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct 60tatattaact tgctatttct agctctaata cnnnnnnnnn nnnnnnnnnn nggtgtttcg 120tcctttccac aag 133232153DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 232acctctagaa aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct 60tattttaact tgctatgctg ttttgtttcc aaaacagcat agctctaaaa cnnnnnnnnn 120nnnnnnnnnn nggtgtttcg tcctttccac aag 153233153DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 233acctctagaa aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct 60tatattaact tgctatgctg tattgtttcc aatacagcat agctctaata cnnnnnnnnn 120nnnnnnnnnn nggtgtttcg tcctttccac aag 15323422DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 234aggccccagt ggctgctctn aa 2223522DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 235acatcaaccg gtggcgcatn at 2223622DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 236aaggtgtggt tccagaaccn ac 2223722DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 237ccatcacatc aaccggtggn ag 2223822DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 238aaacggcaga agctggaggn ta 2223922DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 239ggcagaagct ggaggaggan tt 2224022DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 240ggtgtggttc cagaaccggn tc 2224122DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 241aaccggagga caaagtacan tg 2224222DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 242ttccagaacc ggaggacaan ca 2224322DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 243gtgtggttcc agaaccggan ct 2224422DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 244tccagaaccg gaggacaaan cc 2224522DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 245cagaagctgg aggaggaagn cg 2224622DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 246catcaaccgg tggcgcattn ga 2224722DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 247gcagaagctg gaggaggaan gt 2224822DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 248cctccctccc tggcccaggn gc 2224922DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 249tcatctgtgc ccctccctcn aa 2225022DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 250gggaggacat cgatgtcacn at 2225122DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 251caaacggcag aagctggagn ac 2225222DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 252gggtgggcaa ccacaaaccn ag 2225322DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 253ggtgggcaac cacaaacccn ta 2225422DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 254ggctcccatc acatcaaccn tt 2225522DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 255gaagggcctg agtccgagcn tc 2225622DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 256caaccggtgg cgcattgccn tg 2225722DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 257aggaggaagg gcctgagtcn ca 2225822DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 258agctggagga ggaagggccn ct 2225922DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 259gcattgccac gaagcaggcn cc 2226022DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 260attgccacga agcaggccan cg 2226122DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 261agaaccggag gacaaagtan ga 2226222DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 262tcaaccggtg gcgcattgcn gt 2226322DNAHomo sapiensmodified_base(20)..(20)a, c, t, g, unknown or other 263gaagctggag gaggaagggn gc 22264123DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 264ccaatgggga ggacatcgat gtcacctcca atgactaggg tgggcaacca caaacccacg 60agggcagagt gctgcttgct gctggccagg cccctgcgtg ggcccaagct ggactctggc 120cac 123265121DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 265cgagcagaag aagaagggct cccatcacat caaccggtgg cgcattgcca cgaagcaggc 60caatggggag gacatcgatg tcacctccaa tgactagggt gggcaaccac aaacccacga 120g 121266128DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 266ggaggacaaa gtacaaacgg cagaagctgg aggaggaagg gcctgagtcc gagcagaaga 60agaagggctc ccatcacatc aaccggtggc gcattgccac gaagcaggcc aatggggagg 120acatcgat 128267130DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 267agaagctgga ggaggaaggg cctgagtccg agcagaagaa gaagggctcc catcacatca 60accggtggcg cattgccacg aagcaggcca atggggagga catcgatgtc acctccaatg 120actagggtgg 130268125DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 268cctcagtctt cccatcaggc tctcagctca gcctgagtgt tgaggcccca gtggctgctc 60tgggggcctc ctgagtttct catctgtgcc cctccctccc tggcccaggt gaaggtgtgg 120ttcca 125269129DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 269tcatctgtgc ccctccctcc ctggcccagg tgaaggtgtg gttccagaac cggaggacaa 60agtacaaacg gcagaagctg gaggaggaag ggcctgagtc cgagcagaag aagaagggct 120cccatcaca 129270129DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 270ctccaatgac tagggtgggc aaccacaaac ccacgagggc agagtgctgc ttgctgctgg 60ccaggcccct gcgtgggccc aagctggact ctggccactc cctggccagg ctttggggag 120gcctggagt 129271127DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 271ctgcttgctg ctggccaggc ccctgcgtgg gcccaagctg gactctggcc actccctggc 60caggctttgg ggaggcctgg agtcatggcc ccacagggct tgaagcccgg ggccgccatt 120gacagag 12727225DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 272gaaattaata cgactcacta taggg 25273126DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 273aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac 60ttgctatttc tagctctaaa acaacgacga gcgtgacacc accctatagt gagtcgtatt 120aatttc 126274126DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 274aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac 60ttgctatttc tagctctaaa acgcaacaat taatagactg gacctatagt gagtcgtatt 120aatttc 1262754677DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 275tctttcttgc gctatgacac ttccagcaaa aggtagggcg ggctgcgaga cggcttcccg 60gcgctgcatg caacaccgat gatgcttcga ccccccgaag ctccttcggg gctgcatggg 120cgctccgatg ccgctccagg gcgagcgctg tttaaatagc caggcccccg attgcaaaga 180cattatagcg agctaccaaa gccatattca aacacctaga tcactaccac ttctacacag 240gccactcgag cttgtgatcg cactccgcta agggggcgcc tcttcctctt cgtttcagtc 300acaacccgca aacatgtacc catacgatgt tccagattac gcttcgccga agaaaaagcg 360caaggtcgaa gcgtccgaca agaagtacag catcggcctg gacatcggca ccaactctgt 420gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg 480caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg 540cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa 600gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag 660cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca 720ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta 780ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct 840ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc 900cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt 960cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact 1020gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg 1080cctgttcggc aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt 1140cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga 1200caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct 1260gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc 1320cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa 1380agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa

1440gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat 1500caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga 1560ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct 1620gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa 1680ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc 1740caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg 1800gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac 1860caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga 1920gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa 1980gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa 2040ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga 2100ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga 2160tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct 2220ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct 2280gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata 2340caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg 2400caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct 2460gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca 2520gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg 2580catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc 2640cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa 2700cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct 2760gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct 2820gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta 2880cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt 2940gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt 3000gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa 3060gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt 3120catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga 3180ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat 3240caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg 3300cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc 3360cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta 3420cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta 3480cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga 3540gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa 3600gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa 3660aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag 3720cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag 3780ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa 3840actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa 3900gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat 3960caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc 4020tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct 4080gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca 4140gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt 4200ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa 4260gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac 4320caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta 4380caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta 4440cgagacacgg atcgacctgt ctcagctggg aggcgacagc cccaagaaga agagaaaggt 4500ggaggccagc taaggatccg gcaagactgg ccccgcttgg caacgcaaca gtgagcccct 4560ccctagtgtg tttggggatg tgactatgta ttcgtgtgtt ggccaacggg tcaacccgaa 4620cagattgata cccgccttgg catttcctgt cagaatgtaa cgtcagttga tggtact 46772763150DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 276tctttcttgc gctatgacac ttccagcaaa aggtagggcg ggctgcgaga cggcttcccg 60gcgctgcatg caacaccgat gatgcttcga ccccccgaag ctccttcggg gctgcatggg 120cgctccgatg ccgctccagg gcgagcgctg tttaaatagc caggcccccg attgcaaaga 180cattatagcg agctaccaaa gccatattca aacacctaga tcactaccac ttctacacag 240gccactcgag cttgtgatcg cactccgcta agggggcgcc tcttcctctt cgtttcagtc 300acaacccgca aacatgccta agaagaagag gaaggttaac acgattaaca tcgctaagaa 360cgacttctct gacatcgaac tggctgctat cccgttcaac actctggctg accattacgg 420tgagcgttta gctcgcgaac agttggccct tgagcatgag tcttacgaga tgggtgaagc 480acgcttccgc aagatgtttg agcgtcaact taaagctggt gaggttgcgg ataacgctgc 540cgccaagcct ctcatcacta ccctactccc taagatgatt gcacgcatca acgactggtt 600tgaggaagtg aaagctaagc gcggcaagcg cccgacagcc ttccagttcc tgcaagaaat 660caagccggaa gccgtagcgt acatcaccat taagaccact ctggcttgcc taaccagtgc 720tgacaataca accgttcagg ctgtagcaag cgcaatcggt cgggccattg aggacgaggc 780tcgcttcggt cgtatccgtg accttgaagc taagcacttc aagaaaaacg ttgaggaaca 840actcaacaag cgcgtagggc acgtctacaa gaaagcattt atgcaagttg tcgaggctga 900catgctctct aagggtctac tcggtggcga ggcgtggtct tcgtggcata aggaagactc 960tattcatgta ggagtacgct gcatcgagat gctcattgag tcaaccggaa tggttagctt 1020acaccgccaa aatgctggcg tagtaggtca agactctgag actatcgaac tcgcacctga 1080atacgctgag gctatcgcaa cccgtgcagg tgcgctggct ggcatctctc cgatgttcca 1140accttgcgta gttcctccta agccgtggac tggcattact ggtggtggct attgggctaa 1200cggtcgtcgt cctctggcgc tggtgcgtac tcacagtaag aaagcactga tgcgctacga 1260agacgtttac atgcctgagg tgtacaaagc gattaacatt gcgcaaaaca ccgcatggaa 1320aatcaacaag aaagtcctag cggtcgccaa cgtaatcacc aagtggaagc attgtccggt 1380cgaggacatc cctgcgattg agcgtgaaga actcccgatg aaaccggaag acatcgacat 1440gaatcctgag gctctcaccg cgtggaaacg tgctgccgct gctgtgtacc gcaaggacaa 1500ggctcgcaag tctcgccgta tcagccttga gttcatgctt gagcaagcca ataagtttgc 1560taaccataag gccatctggt tcccttacaa catggactgg cgcggtcgtg tttacgctgt 1620gtcaatgttc aacccgcaag gtaacgatat gaccaaagga ctgcttacgc tggcgaaagg 1680taaaccaatc ggtaaggaag gttactactg gctgaaaatc cacggtgcaa actgtgcggg 1740tgtcgacaag gttccgttcc ctgagcgcat caagttcatt gaggaaaacc acgagaacat 1800catggcttgc gctaagtctc cactggagaa cacttggtgg gctgagcaag attctccgtt 1860ctgcttcctt gcgttctgct ttgagtacgc tggggtacag caccacggcc tgagctataa 1920ctgctccctt ccgctggcgt ttgacgggtc ttgctctggc atccagcact tctccgcgat 1980gctccgagat gaggtaggtg gtcgcgcggt taacttgctt cctagtgaaa ccgttcagga 2040catctacggg attgttgcta agaaagtcaa cgagattcta caagcagacg caatcaatgg 2100gaccgataac gaagtagtta ccgtgaccga tgagaacact ggtgaaatct ctgagaaagt 2160caagctgggc actaaggcac tggctggtca atggctggct tacggtgtta ctcgcagtgt 2220gactaagcgt tcagtcatga cgctggctta cgggtccaaa gagttcggct tccgtcaaca 2280agtgctggaa gataccattc agccagctat tgattccggc aagggtctga tgttcactca 2340gccgaatcag gctgctggat acatggctaa gctgatttgg gaatctgtga gcgtgacggt 2400ggtagctgcg gttgaagcaa tgaactggct taagtctgct gctaagctgc tggctgctga 2460ggtcaaagat aagaagactg gagagattct tcgcaagcgt tgcgctgtgc attgggtaac 2520tcctgatggt ttccctgtgt ggcaggaata caagaagcct attcagacgc gcttgaacct 2580gatgttcctc ggtcagttcc gcttacagcc taccattaac accaacaaag atagcgagat 2640tgatgcacac aaacaggagt ctggtatcgc tcctaacttt gtacacagcc aagacggtag 2700ccaccttcgt aagactgtag tgtgggcaca cgagaagtac ggaatcgaat cttttgcact 2760gattcacgac tccttcggta cgattccggc tgacgctgcg aacctgttca aagcagtgcg 2820cgaaactatg gttgacacat atgagtcttg tgatgtactg gctgatttct acgaccagtt 2880cgctgaccag ttgcacgagt ctcaattgga caaaatgcca gcacttccgg ctaaaggtaa 2940cttgaacctc cgtgacatct tagagtcgga cttcgcgttc gcgtaaggat ccggcaagac 3000tggccccgct tggcaacgca acagtgagcc cctccctagt gtgtttgggg atgtgactat 3060gtattcgtgt gttggccaac gggtcaaccc gaacagattg atacccgcct tggcatttcc 3120tgtcagaatg taacgtcagt tgatggtact 3150277125DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 277gaaattaata cgactcacta tannnnnnnn nnnnnnnnnn nngttttaga gctagaaata 60gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 120ttttt 1252788452DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 278tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat 60ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagattat caaaaaggat 120cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 180gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 240tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 300gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 360agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 420tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 480agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 540gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 600catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 660ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 720atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 780tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 840cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 900cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 960atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 1020aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 1080ttgaagcatt tatcagggtt attgtctcat gaccaaaatc ccttaacgtg agttttcgtt 1140ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 1200gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 1260ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 1320aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 1380gcctacatac ctcgctctgc taatcctgtt accagtggct gttgccagtg gcgataagtc 1440gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 1500aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 1560cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 1620tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 1680ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 1740atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 1800cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 1860ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 1920gcgcagcgag tcagtgagcg aggaagcggt cgctgaggct tgacatgatt ggtgcgtatg 1980tttgtatgaa gctacaggac tgatttggcg ggctatgagg gcgggggaag ctctggaagg 2040gccgcgatgg ggcgcgcggc gtccagaagg cgccatacgg cccgctggcg gcacccatcc 2100ggtataaaag cccgcgaccc cgaacggtga cctccacttt cagcgacaaa cgagcactta 2160tacatacgcg actattctgc cgctatacat aaccactcag ctagcttaag atcccatcaa 2220gcttgcatgc cgggcgcgcc agaaggagcg cagccaaacc aggatgatgt ttgatggggt 2280atttgagcac ttgcaaccct tatccggaag ccccctggcc cacaaaggct aggcgccaat 2340gcaagcagtt cgcatgcagc ccctggagcg gtgccctcct gataaaccgg ccagggggcc 2400tatgttcttt acttttttac aagagaagtc actcaacatc ttaaaatggc caggtgagtc 2460gacgagcaag cccggcggat caggcagcgt gcttgcagat ttgacttgca acgcccgcat 2520tgtgtcgacg aaggcttttg gctcctctgt cgctgtctca agcagcatct aaccctgcgt 2580cgccgtttcc atttgcagga gattcgaggt accatgtacc catacgatgt tccagattac 2640gcttcgccga agaaaaagcg caaggtcgaa gcgtccgaca agaagtacag catcggcctg 2700gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc 2760aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga 2820gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga 2880agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag 2940atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag 3000gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac 3060gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc 3120gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg 3180atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg 3240cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag 3300gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg 3360cccggcgaga agaagaatgg cctgttcggc aacctgattg ccctgagcct gggcctgacc 3420cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac 3480acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg 3540tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac 3600accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac 3660caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag 3720attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag 3780gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg 3840ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc 3900atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt 3960tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc 4020tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc 4080gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag 4140agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc 4200aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac 4260gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg 4320gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc 4380aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc 4440tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat 4500gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga 4560gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag 4620cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc 4680atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc 4740aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag 4800aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc 4860agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa 4920gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc 4980acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa 5040gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag 5100aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac 5160atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac 5220gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac 5280gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc 5340aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc 5400gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag 5460cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg 5520atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat 5580ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg 5640aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg 5700tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc 5760ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag 5820attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc 5880ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg 5940ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct 6000atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag 6060aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg 6120gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg 6180gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa 6240gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc 6300cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc 6360tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc 6420gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc 6480atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa 6540gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc 6600atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc 6660accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac 6720cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacagc 6780cccaagaaga agagaaaggt ggaggccagc taacatatga ttcgaatgtc tttcttgcgc 6840tatgacactt ccagcaaaag gtagggcggg ctgcgagacg gcttcccggc gctgcatgca 6900acaccgatga tgcttcgacc ccccgaagct ccttcggggc tgcatgggcg ctccgatgcc 6960gctccagggc gagcgctgtt taaatagcca ggcccccgat tgcaaagaca ttatagcgag 7020ctaccaaagc catattcaaa cacctagatc actaccactt ctacacaggc cactcgagct 7080tgtgatcgca ctccgctaag ggggcgcctc ttcctcttcg tttcagtcac aacccgcaaa 7140catgacacaa gaatccctgt tacttctcga ccgtattgat tcggatgatt cctacgcgag 7200cctgcggaac gaccaggaat tctgggaggt gagtcgacga gcaagcccgg cggatcaggc 7260agcgtgcttg cagatttgac ttgcaacgcc cgcattgtgt cgacgaaggc ttttggctcc 7320tctgtcgctg tctcaagcag catctaaccc tgcgtcgccg tttccatttg cagccgctgg 7380cccgccgagc cctggaggag ctcgggctgc cggtgccgcc ggtgctgcgg gtgcccggcg 7440agagcaccaa ccccgtactg gtcggcgagc ccggcccggt gatcaagctg ttcggcgagc 7500actggtgcgg tccggagagc ctcgcgtcgg agtcggaggc gtacgcggtc ctggcggacg 7560ccccggtgcc ggtgccccgc ctcctcggcc gcggcgagct gcggcccggc accggagcct 7620ggccgtggcc ctacctggtg atgagccgga tgaccggcac cacctggcgg tccgcgatgg 7680acggcacgac cgaccggaac gcgctgctcg ccctggcccg cgaactcggc cgggtgctcg 7740gccggctgca cagggtgccg ctgaccggga acaccgtgct caccccccat tccgaggtct 7800tcccggaact gctgcgggaa cgccgcgcgg cgaccgtcga ggaccaccgc gggtggggct 7860acctctcgcc ccggctgctg gaccgcctgg aggactggct gccggacgtg gacacgctgc 7920tggccggccg cgaaccccgg ttcgtccacg gcgacctgca cgggaccaac atcttcgtgg 7980acctggccgc gaccgaggtc accgggatcg tcgacttcac cgacgtctat gcgggagact 8040cccgctacag cctggtgcaa ctgcatctca acgccttccg gggcgaccgc gagatcctgg 8100ccgcgctgct cgacggggcg cagtggaagc ggaccgagga cttcgcccgc gaactgctcg 8160ccttcacctt cctgcacgac ttcgaggtgt

tcgaggagac cccgctggat ctctccggct 8220tcaccgatcc ggaggaactg gcgcagttcc tctgggggcc gccggacacc gcccccggcg 8280cctgataagg atccggcaag actggccccg cttggcaacg caacagtgag cccctcccta 8340gtgtgtttgg ggatgtgact atgtattcgt gtgttggcca acgggtcaac ccgaacagat 8400tgatacccgc cttggcattt cctgtcagaa tgtaacgtca gttgatggta ct 8452279102DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 279gttttagagc tatgctgttt tgaatggtcc caaaacggaa gggcctgagt ccgagcagaa 60gaagaagttt tagagctatg ctgttttgaa tggtcccaaa ac 102280100DNAHomo sapiens 280cggaggacaa agtacaaacg gcagaagctg gaggaggaag ggcctgagtc cgagcagaag 60aagaagggct cccatcacat caaccggtgg cgcattgcca 10028150DNAHomo sapiens 281agctggagga ggaagggcct gagtccgagc agaagaagaa gggctcccac 5028230RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 282gaguccgagc agaagaagaa guuuuagagc 3028349DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 283agctggagga ggaagggcct gagtccgagc agaagagaag ggctcccat 4928453DNAHomo sapiens 284ctggaggagg aagggcctga gtccgagcag aagaagaagg gctcccatca cat 5328552DNAHomo sapiens 285ctggaggagg aagggcctga gtccgagcag aagagaaggg ctcccatcac at 5228654DNAHomo sapiens 286ctggaggagg aagggcctga gtccgagcag aagaaagaag ggctcccatc acat 5428750DNAHomo sapiens 287ctggaggagg aagggcctga gtccgagcag aagaagggct cccatcacat 5028847DNAHomo sapiens 288ctggaggagg aagggcctga gcccgagcag aagggctccc atcacat 4728966DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 289nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggctagtc 60cguuuu 6629020RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 290gaguccgagc agaagaagaa 2029120RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 291gacaucgaug uccuccccau 2029220RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 292gucaccucca augacuaggg 2029320RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 293auuggguguu cagggcagag 2029420RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 294guggcgagag gggccgagau 2029520RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 295ggggccgaga uuggguguuc 2029620RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 296gugccauuag cuaaaugcau 2029720RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 297guaccaccca caggugccag 2029820RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 298gaaagccucu gggccaggaa 2029948DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 299ctggaggagg aagggcctga gtccgagcag aagaagaagg gctcccat 4830020RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 300gaguccgagc agaagaagau 2030120RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 301gaguccgagc agaagaagua 2030220RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 302gaguccgagc agaagaacaa 2030320RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 303gaguccgagc agaagaugaa 2030420RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 304gaguccgagc agaaguagaa 2030520RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 305gaguccgagc agaugaagaa 2030620RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 306gaguccgagc acaagaagaa 2030720RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 307gaguccgagg agaagaagaa 2030820RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 308gaguccgugc agaagaagaa 2030920RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 309gagucggagc agaagaagaa 2031020RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 310gagaccgagc agaagaagaa 2031124DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 311aatgacaagc ttgctagcgg tggg 2431239DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 312aaaacggaag ggcctgagtc cgagcagaag aagaagttt 3931339DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 313aaacaggggc cgagattggg tgttcagggc agaggtttt 3931438DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 314aaaacggaag ggcctgagtc cgagcagaag aagaagtt 3831540DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 315aacggaggga ggggcacaga tgagaaactc agggttttag 4031638DNAHomo sapiens 316agcccttctt cttctgctcg gactcaggcc cttcctcc 3831740DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 317cagggaggga ggggcacaga tgagaaactc aggaggcccc 4031880DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 318ggcaatgcgc caccggttga tgtgatggga gcccttctag gaggccccca gagcagccac 60tggggcctca acactcaggc 8031923DNAHomo sapiens 319gtcacctcca atgactaggg tgg 2332025DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 320caccgnnnnn nnnnnnnnnn nnnnn 2532125DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 321aaacnnnnnn nnnnnnnnnn nnnnc 2532233DNAHomo sapiens 322catcgatgtc ctccccattg gcctgcttcg tgg 3332333DNAHomo sapiens 323ttcgtggcaa tgcgccaccg gttgatgtga tgg 3332433DNAHomo sapiens 324tcgtggcaat gcgccaccgg ttgatgtgat ggg 3332533DNAHomo sapiens 325tccagcttct gccgtttgta ctttgtcctc cgg 3332633DNAHomo sapiens 326ggagggaggg gcacagatga gaaactcagg agg 3332733DNAHomo sapiens 327aggggccgag attgggtgtt cagggcagag agg 3332854DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 328aacaccgggt cttcgagaag acctgtttta gagctagaaa tagcaagtta aaat 5432954DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 329caaaacgggt cttcgagaag acgttttaga gctatgctgt tttgaatggt ccca 5433033DNAMus musculus 330caagcactga gtgccattag ctaaatgcat agg 3333133DNAMus musculus 331aatgcatagg gtaccaccca caggtgccag ggg 3333233DNAMus musculus 332acacacatgg gaaagcctct gggccaggaa agg 3333337DNAHomo sapiens 333ggaggaggta gtatacagaa acacagagaa gtagaat 3733437DNAHomo sapiens 334agaatgtaga ggagtcacag aaactcagca ctagaaa 3733598DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 335ggacgaaaca ccggaaccat tcaaaacagc atagcaagtt aaaataaggc tagtccgtta 60tcaacttgaa aaagtggcac cgagtcggtg cttttttt 98336186DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 336ggacgaaaca ccggtagtat taagtattgt tttatggctg ataaatttct ttgaatttct 60ccttgattat ttgttataaa agttataaaa taatcttgtt ggaaccattc aaaacagcat 120agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 180tttttt 18633795DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 337gggttttaga gctatgctgt tttgaatggt cccaaaacgg gtcttcgaga agacgtttta 60gagctatgct gttttgaatg gtcccaaaac ttttt 9533836DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 338aaacnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngt 3633936DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 339taaaacnnnn nnnnnnnnnn nnnnnnnnnn nnnnnn 3634084DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 340gtggaaagga cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag 60ttaaaataag gctagtccgt tttt 8434124DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 341caccgnnnnn nnnnnnnnnn nnnn 2434224DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 342aaacnnnnnn nnnnnnnnnn nnnc 2434388DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 343gttttagagc tatgctgttt tgaatggtcc caaaactgag accaaaggtc tcgttttaga 60gctatgctgt tttgaatggt cccaaaac 8834435DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 344aaacggaagg gcctgagtcc gagcagaaga agaag 3534535DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 345aaaacttctt cttctgctcg gactcaggcc cttcc 3534646RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 346nnnnnnnnnn nnnnnnnnng uuauuguacu cucaagauuu auuuuu 4634791RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 347guuacuuaaa ucuugcagaa gcuacaaaga uaaggcuuca ugccgaaauc aacacccugu 60cauuuuaugg caggguguuu ucguuauuua a 9134870DNAHomo sapiens 348ttttctagtg ctgagtttct gtgactcctc tacattctac ttctctgtgt ttctgtatac 60tacctcctcc 70349122DNAHomo sapiens 349ggaggaaggg cctgagtccg agcagaagaa gaagggctcc catcacatca accggtggcg 60cattgccacg aagcaggcca atggggagga catcgatgtc acctccaatg actagggtgg 120gc 12235048RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 350acnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnguuuuaga gcuaugcu 4835167DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 351agcauagcaa guuaaaauaa ggctaguccg uuaucaacuu gaaaaagugg caccgagucg 60gugcuuu 6735262RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 352nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cg 6235373DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 353tgaatggtcc caaaacggaa gggcctgagt ccgagcagaa gaagaagttt tagagctatg 60ctgttttgaa tgg 7335469DNAHomo sapiens 354ctggtcttcc acctctctgc cctgaacacc caatctcggc ccctctcgcc accctcctgc 60atttctgtt 69355138DNAMus musculus 355acccaagcac tgagtgccat tagctaaatg catagggtac cacccacagg tgccaggggc 60ctttcccaaa gttcccagcc ccttctccaa cctttcctgg cccagaggct ttcccatgtg 120tgtggctgga ccctttga 13835620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 356gtgctttgca gaggcctacc 2035720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 357cctggagcgc atgcagtagt 2035822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 358accttctgtg tttccaccat tc 2235920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 359ttggggagtg cacagacttc 2036020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 360ggctccctgg gttcaaagta 2036121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 361agaggggtct ggatgtcgta a 2136230DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 362tagctctaaa acttcttctt ctgctcggac 3036330DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 363ctagccttat tttaacttgc tatgctgttt 3036499RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 364nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuu 9936512DNAHomo sapiens 365tagcgggtaa gc 1236612DNAHomo sapiens 366tcggtgacat gt 1236712DNAHomo sapiens 367actccccgta gg 1236812DNAHomo sapiens 368actgcgtgtt aa 1236912DNAHomo sapiens 369acgtcgcctg at 1237012DNAHomo sapiens 370taggtcgacc ag 1237112DNAHomo sapiens 371ggcgttaatg at 1237212DNAHomo sapiens 372tgtcgcatgt ta 1237312DNAHomo sapiens 373atggaaacgc at 1237412DNAHomo sapiens 374gccgaattcc tc 1237512DNAHomo sapiens 375gcatggtacg ga 1237612DNAHomo sapiens 376cggtactctt ac 1237712DNAHomo sapiens 377gcctgtgccg ta 1237812DNAHomo sapiens 378tacggtaagt cg 1237912DNAHomo sapiens 379cacgaaatta cc 1238012DNAHomo sapiens 380aaccaagata cg 1238112DNAHomo sapiens 381gagtcgatac gc 1238212DNAHomo sapiens 382gtctcacgat cg 1238312DNAHomo sapiens 383tcgtcgggtg ca 1238412DNAHomo sapiens 384actccgtagt ga 1238512DNAHomo sapiens 385caggacgtcc gt 1238612DNAHomo sapiens 386tcgtatccct ac 1238712DNAHomo sapiens 387tttcaaggcc gg 1238812DNAHomo sapiens 388cgccggtgga at 1238912DNAHomo sapiens 389gaacccgtcc ta 1239012DNAHomo sapiens 390gattcatcag cg 1239112DNAHomo sapiens 391acaccggtct tc 1239212DNAHomo sapiens 392atcgtgccct aa 1239312DNAHomo sapiens 393gcgtcaatgt tc 1239412DNAHomo sapiens 394ctccgtatct cg 1239512DNAHomo sapiens 395ccgattcctt cg

1239612DNAHomo sapiens 396tgcgcctcca gt 1239712DNAHomo sapiens 397taacgtcgga gc 1239812DNAHomo sapiens 398aaggtcgccc at 1239912DNAHomo sapiens 399gtcggggact at 1240012DNAHomo sapiens 400ttcgagcgat tt 1240112DNAHomo sapiens 401tgagtcgtcg ag 1240212DNAHomo sapiens 402tttacgcaga gg 1240312DNAHomo sapiens 403aggaagtatc gc 1240412DNAHomo sapiens 404actcgatacc at 1240512DNAHomo sapiens 405cgctacatag ca 1240612DNAHomo sapiens 406ttcataaccg gc 1240712DNAHomo sapiens 407ccaaacggtt aa 1240812DNAHomo sapiens 408cgattccttc gt 1240912DNAHomo sapiens 409cgtcatgaat aa 1241012DNAHomo sapiens 410agtggcgatg ac 1241112DNAHomo sapiens 411cccctacggc ac 1241212DNAHomo sapiens 412gccaacccgc ac 1241312DNAHomo sapiens 413tgggacaccg gt 1241412DNAHomo sapiens 414ttgactgcgg cg 1241512DNAHomo sapiens 415actatgcgta gg 1241612DNAHomo sapiens 416tcacccaaag cg 1241712DNAHomo sapiens 417gcaggacgtc cg 1241812DNAHomo sapiens 418acaccgaaaa cg 1241912DNAHomo sapiens 419cggtgtattg ag 1242012DNAHomo sapiens 420cacgaggtat gc 1242112DNAHomo sapiens 421taaagcgacc cg 1242212DNAHomo sapiens 422cttagtcggc ca 1242312DNAHomo sapiens 423cgaaaacgtg gc 1242412DNAHomo sapiens 424cgtgccctga ac 1242512DNAHomo sapiens 425tttaccatcg aa 1242612DNAHomo sapiens 426cgtagccatg tt 1242712DNAHomo sapiens 427cccaaacggt ta 1242812DNAHomo sapiens 428gcgttatcag aa 1242912DNAHomo sapiens 429tcgatggtaa ac 1243012DNAHomo sapiens 430cgactttttg ca 1243112DNAHomo sapiens 431tcgacgactc ac 1243212DNAHomo sapiens 432acgcgtcaga ta 1243312DNAHomo sapiens 433cgtacggcac ag 1243412DNAHomo sapiens 434ctatgccgtg ca 1243512DNAHomo sapiens 435cgcgtcagat at 1243612DNAHomo sapiens 436aagatcggta gc 1243712DNAHomo sapiens 437cttcgcaagg ag 1243812DNAHomo sapiens 438gtcgtggact ac 1243912DNAHomo sapiens 439ggtcgtcatc aa 1244012DNAHomo sapiens 440gttaacagcg tg 1244112DNAHomo sapiens 441tagctaaccg tt 1244212DNAHomo sapiens 442agtaaaggcg ct 1244312DNAHomo sapiens 443ggtaatttcg tg 1244469RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 444gucaccucca augacuaggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuuuuuu 6944569RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 445gacaucgaug uccuccccau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuuuuuu 6944669RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 446gaguccgagc agaagaagaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuuuuuu 6944769RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 447ggggccgaga uuggguguuc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuuuuuu 6944869RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 448guggcgagag gggccgagau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuuuuuu 6944976RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 449gucaccucca augacuaggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu 7645076RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 450gacaucgaug uccuccccau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu 7645176RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 451gaguccgagc agaagaagaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu 7645276RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 452ggggccgaga uuggguguuc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu 7645376RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 453guggcgagag gggccgagau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucauu uuuuuu 7645488RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 454gucaccucca augacuaggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu 8845588RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 455gacaucgaug uccuccccau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu 8845688RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 456gaguccgagc agaagaagaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu 8845788RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 457ggggccgaga uuggguguuc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu 8845888RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 458guggcgagag gggccgagau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu guuuuuuu 88459103RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 459gucaccucca augacuaggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103460103RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 460gacaucgaug uccuccccau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103461103RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 461gaguccgagc agaagaagaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103462103RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 462ggggccgaga uuggguguuc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103463103RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 463guggcgagag gggccgagau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103464120DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 464gtggaaagga cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag 60ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 12046540DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 465tcggtgcgct ggttgatttc ttcttgcgct tttttggctt 4046626RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 466gauuucuucu ugcgcuuuuu guuuua 2646726DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 467tgatttcttc ttgcgctttt tnnnnn 2646826DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 468tgatttcttc ttgcgctttt ntggct 2646926DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 469tnatttcttc ttgcgctttt ttggct 2647023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 470gatttcttct tgcgcttttt tgg 2347134DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 471tcc atc cgt aca acc cac aac cct gct agt gag c 34Ser Ile Arg Thr Thr His Asn Pro Ala Ser Glu 1 5 10 47211PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 472Ser Ile Arg Thr Thr His Asn Pro Ala Ser Glu1 5 10 47334DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 473tcc atc gca aca acc cac aac cct gct agt gag c 34Ser Ile Ala Thr Thr His Asn Pro Ala Ser Glu 1 5 10 47411PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 474Ser Ile Ala Thr Thr His Asn Pro Ala Ser Glu 1 5 10 47534DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 475tca atc cgt aca acc cac aac cct gct agt gag c 34Ser Ile Arg Thr Thr His Asn Pro Ala Ser Glu 1 5 10 47642DNAHomo sapiensCDS(1)..(36) 476caa ttg aat tta aaa gaa acc gat acc gtt ttg gtt taagga 42Gln Leu Asn Leu Lys Glu Thr Asp Thr Val Leu Val 1 5 10 47712PRTHomo sapiens 477Gln Leu Asn Leu Lys Glu Thr Asp Thr Val Leu Val 1 5 10 47842DNAHomo sapiensCDS(1)..(42) 478caa ttg aat tta aaa gaa acc gat acc gtt tac gaa att gga 42Gln Leu Asn Leu Lys Glu Thr Asp Thr Val Tyr Glu Ile Gly 1 5 10 47914PRTHomo sapiens 479Gln Leu Asn Leu Lys Glu Thr Asp Thr Val Tyr Glu Ile Gly 1 5 10 48034DNAHomo sapiensCDS(2)..(34) 480t cct aaa aaa ccg aac tcc gcg ctg cgt aaa gta 34Pro Lys Lys Pro Asn Ser Ala Leu Arg Lys Val 1 5 10 48111PRTHomo sapiens 481Pro Lys Lys Pro Asn Ser Ala Leu Arg Lys Val 1 5 10 48234DNAHomo sapiensCDS(2)..(34) 482t cct aca aaa ccg aac tcc gcg ctg cgt aaa gta 34Pro Thr Lys Pro Asn Ser Ala Leu Arg Lys Val 1 5 10 48311PRTHomo sapiens 483Pro Thr Lys Pro Asn Ser Ala Leu Arg Lys Val 1 5 10 48433DNAHomo sapiens 484tgcgctggtt gatttcttct tgcgcttttt tgg 3348533DNAHomo sapiens 485tacgctggtt gatttcttct tgcgcttttt ttg 3348627DNAHomo sapiens 486ggagggtttt atggggaaag gccattg 2748729DNAHomo sapiens 487gtaaaaaaga agactagaaa ttttgatac 2948846DNAHomo sapiens 488ggagggtttt atggggaaag gcaaagaaga ctagaaattt tgatac 4648927DNAHomo sapiens 489aggtgaagca taatgtctca aaaaata 2749029DNAHomo sapiens 490attttattaa tacaaatcag tgaaatcat 2949146DNAHomo sapiens 491aggtgaagca taatgtctca aaattaatac aaatcagtga aatcat 4649236DNAHomo sapiensCDS(1)..(36) 492aat tta aaa gaa acc gat acc gtt tac gaa att gga 36Asn Leu Lys Glu Thr Asp Thr Val Tyr Glu Ile Gly 1 5 10 49312PRTHomo sapiens 493Asn Leu Lys Glu Thr Asp Thr Val Tyr Glu Ile Gly 1 5 10 49436DNAHomo sapiensCDS(1)..(30) 494aat tta aaa gaa acc gat acc gtt ttg gtt taagga 36Asn Leu Lys Glu Thr Asp Thr Val Leu Val 1 5 10 49510PRTHomo sapiens 495Asn Leu Lys Glu Thr Asp Thr Val Leu Val 1 5 10 49636DNAHomo sapiensCDS(1)..(36) 496tgg gat cca aaa aaa tat ggt ggt ttt gat agt cca 36Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro 1 5 10 49712PRTHomo sapiens 497Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro 1 5 10 49836DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 498tgg gat cca aaa aaa tat tgt ggt ttt gat agt cca 36Trp Asp Pro Lys Lys Tyr Cys Gly Phe Asp Ser Pro 1 5 10 49912PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 499Trp Asp Pro Lys Lys Tyr Cys Gly Phe Asp Ser Pro 1 5 10 50035DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 500aaactacttt acgcagcgcg gagttcggtt ttttg 355014104DNAHomo sapiensCDS(1)..(4104) 501atg gac aag aag tac agc atc ggc ctg gac atc ggc acc aac tct gtg 48Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 ggc tgg gcc gtg atc acc gac gag tac aag gtg ccc agc aag aaa ttc 96Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 aag gtg ctg ggc aac acc gac cgg cac agc atc aag aag aac ctg atc 144Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 gga gcc ctg ctg ttc gac agc ggc gaa aca gcc gag gcc acc cgg ctg 192Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 aag aga acc gcc aga aga aga tac acc aga cgg aag aac cgg atc tgc 240Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 tat ctg caa gag atc ttc agc aac gag atg gcc aag gtg gac gac agc 288Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 ttc ttc cac aga ctg gaa gag tcc ttc ctg gtg gaa gag gat aag aag 336Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 cac gag cgg cac ccc atc ttc ggc aac atc gtg gac gag gtg gcc tac 384His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 cac gag aag tac ccc acc atc tac cac ctg aga

aag aaa ctg gtg gac 432His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 agc acc gac aag gcc gac ctg cgg ctg atc tat ctg gcc ctg gcc cac 480Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 atg atc aag ttc cgg ggc cac ttc ctg atc gag ggc gac ctg aac ccc 528Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 gac aac agc gac gtg gac aag ctg ttc atc cag ctg gtg cag acc tac 576Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 aac cag ctg ttc gag gaa aac ccc atc aac gcc agc ggc gtg gac gcc 624Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 aag gcc atc ctg tct gcc aga ctg agc aag agc aga cgg ctg gaa aat 672Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 ctg atc gcc cag ctg ccc ggc gag aag aag aat ggc ctg ttc ggc aac 720Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 ctg att gcc ctg agc ctg ggc ctg acc ccc aac ttc aag agc aac ttc 768Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 gac ctg gcc gag gat gcc aaa ctg cag ctg agc aag gac acc tac gac 816Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 gac gac ctg gac aac ctg ctg gcc cag atc ggc gac cag tac gcc gac 864Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 ctg ttt ctg gcc gcc aag aac ctg tcc gac gcc atc ctg ctg agc gac 912Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 atc ctg aga gtg aac acc gag atc acc aag gcc ccc ctg agc gcc tct 960Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 atg atc aag aga tac gac gag cac cac cag gac ctg acc ctg ctg aaa 1008Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 gct ctc gtg cgg cag cag ctg cct gag aag tac aaa gag att ttc ttc 1056Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 gac cag agc aag aac ggc tac gcc ggc tac att gac ggc gga gcc agc 1104Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 cag gaa gag ttc tac aag ttc atc aag ccc atc ctg gaa aag atg gac 1152Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 ggc acc gag gaa ctg ctc gtg aag ctg aac aga gag gac ctg ctg cgg 1200Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 aag cag cgg acc ttc gac aac ggc agc atc ccc cac cag atc cac ctg 1248Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 gga gag ctg cac gcc att ctg cgg cgg cag gaa gat ttt tac cca ttc 1296Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 ctg aag gac aac cgg gaa aag atc gag aag atc ctg acc ttc cgc atc 1344Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 ccc tac tac gtg ggc cct ctg gcc agg gga aac agc aga ttc gcc tgg 1392Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 atg acc aga aag agc gag gaa acc atc acc ccc tgg aac ttc gag gaa 1440Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 gtg gtg gac aag ggc gct tcc gcc cag agc ttc atc gag cgg atg acc 1488Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 aac ttc gat aag aac ctg ccc aac gag aag gtg ctg ccc aag cac agc 1536Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 ctg ctg tac gag tac ttc acc gtg tat aac gag ctg acc aaa gtg aaa 1584Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 tac gtg acc gag gga atg aga aag ccc gcc ttc ctg agc ggc gag cag 1632Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 aaa aag gcc atc gtg gac ctg ctg ttc aag acc aac cgg aaa gtg acc 1680Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 gtg aag cag ctg aaa gag gac tac ttc aag aaa atc gag tgc ttc gac 1728Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 tcc gtg gaa atc tcc ggc gtg gaa gat cgg ttc aac gcc tcc ctg ggc 1776Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 aca tac cac gat ctg ctg aaa att atc aag gac aag gac ttc ctg gac 1824Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 aat gag gaa aac gag gac att ctg gaa gat atc gtg ctg acc ctg aca 1872Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 ctg ttt gag gac aga gag atg atc gag gaa cgg ctg aaa acc tat gcc 1920Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 cac ctg ttc gac gac aaa gtg atg aag cag ctg aag cgg cgg aga tac 1968His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 acc ggc tgg ggc agg ctg agc cgg aag ctg atc aac ggc atc cgg gac 2016Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 aag cag tcc ggc aag aca atc ctg gat ttc ctg aag tcc gac ggc ttc 2064Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 gcc aac aga aac ttc atg cag ctg atc cac gac gac agc ctg acc ttt 2112Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 aaa gag gac atc cag aaa gcc cag gtg tcc ggc cag ggc gat agc ctg 2160Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 cac gag cac att gcc aat ctg gcc ggc agc ccc gcc att aag aag ggc 2208His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 atc ctg cag aca gtg aag gtg gtg gac gag ctc gtg aaa gtg atg ggc 2256Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 cgg cac aag ccc gag aac atc gtg atc gcc atg gcc aga gag aac cag 2304Arg His Lys Pro Glu Asn Ile Val Ile Ala Met Ala Arg Glu Asn Gln 755 760 765 acc acc cag aag gga cag aag aac agc cgc gag aga atg aag cgg atc 2352Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 gaa gag ggc atc aaa gag ctg ggc agc cag atc ctg aaa gaa cac ccc 2400Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 gtg gaa aac acc cag ctg cag aac gag aag ctg tac ctg tac tac ctg 2448Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 cag aat ggg cgg gat atg tac gtg gac cag gaa ctg gac atc aac cgg 2496Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 ctg tcc gac tac gat gtg gac gcc atc gtg cct cag agc ttt ctg aag 2544Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 gac gac tcc atc gac gcc aag gtg ctg acc aga agc gac aag gcc cgg 2592Asp Asp Ser Ile Asp Ala Lys Val Leu Thr Arg Ser Asp Lys Ala Arg 850 855 860 ggc aag agc gac aac gtg ccc tcc gaa gag gtc gtg aag aag atg aag 2640Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 aac tac tgg cgg cag ctg ctg aac gcc aag ctg att acc cag aga aag 2688Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 ttc gac aat ctg acc aag gcc gag aga ggc ggc ctg agc gaa ctg gat 2736Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 aag gcc ggc ttc atc aag aga cag ctg gtg gaa acc cgg cag atc aca 2784Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 aag cac gtg gca cag atc ctg gac tcc cgg atg aac act aag tac gac 2832Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 gag aat gac aag ctg atc cgg gaa gtg aaa gtg atc acc ctg aag tcc 2880Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 aag ctg gtg tcc gat ttc cgg aag gat ttc cag ttt tac aaa gtg cgc 2928Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 gag atc aac aac tac cac cac gcc cac gcc gcc tac ctg aac gcc gtc 2976Glu Ile Asn Asn Tyr His His Ala His Ala Ala Tyr Leu Asn Ala Val 980 985 990 gtg gga acc gcc ctg atc aaa aag tac cct aag ctg gaa agc gag ttc 3024Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 gtg tac ggc gac tac aag gtg tac gac gtg cgg aag atg atc gcc 3069Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 aag agc gag cag gaa atc ggc aag gct acc gcc aag tac ttc ttc 3114Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 tac agc aac atc atg aac ttt ttc aag acc gag att acc ctg gcc 3159Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 aac ggc gag atc cgg aag cgg cct ctg atc gag aca aac ggc gaa 3204Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 acc ggg gag atc gtg tgg gat aag ggc cgg gat ttt gcc acc gtg 3249Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 cgg aaa gtg ctg agc atg ccc caa gtg aat atc gtg aaa aag acc 3294Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 gag gtg cag aca ggc ggc ttc agc aaa gag tct atc ctg ccc aag 3339Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 agg aac agc gat aag ctg atc gcc aga aag aag gac tgg gac cct 3384Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 aag aag tac ggc ggc ttc gac agc ccc acc gtg gcc tat tct gtg 3429Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 ctg gtg gtg gcc aaa gtg gaa aag ggc aag tcc aag aaa ctg aag 3474Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 agt gtg aaa gag ctg ctg ggg atc acc atc atg gaa aga agc agc 3519Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 ttc gag aag aat ccc atc gac ttt ctg gaa gcc aag ggc tac aaa 3564Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 gaa gtg aaa aag gac ctg atc atc aag ctg cct aag tac tcc ctg 3609Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 ttc gag ctg gaa aac ggc cgg aag aga atg ctg gcc tct gcc ggc 3654Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 gaa ctg cag aag gga aac gaa ctg gcc ctg ccc tcc aaa tat gtg 3699Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 aac ttc ctg tac ctg gcc agc cac tat gag aag ctg aag ggc tcc 3744Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 ccc gag gat aat gag cag aaa cag ctg ttt gtg gaa cag cac aag 3789Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 cac tac ctg gac gag atc atc gag cag atc agc gag ttc tcc aag 3834His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 aga gtg atc ctg gcc gac gct aat ctg gac aaa gtg ctg tcc gcc 3879Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 tac aac aag cac cgg gat aag ccc atc aga gag cag gcc gag aat 3924Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 atc atc cac ctg ttt acc ctg acc aat ctg gga gcc cct gcc gcc 3969Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 ttc aag tac ttt gac acc acc atc gac cgg aag agg tac acc agc 4014Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 acc aaa gag gtg ctg gac gcc acc ctg atc cac cag agc atc acc 4059Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 ggc ctg tac gag aca cgg atc gac ctg tct cag ctg gga ggc gac 4104Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 5021368PRTHomo sapiens 502Met 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 Ala 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 Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Ala Lys Val Leu Thr Arg Ser Asp Lys Ala 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 Ala 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 50315DNAHomo sapiens 503cagaagaaga agggc 1550451DNAHomo sapiens 504ccaatgggga ggacatcgat gtcacctcca atgactaggg tggtgggcaa c 5150515DNAHomo sapiens 505ctctggccac tccct 1550652DNAHomo sapiens 506acatcgatgt cacctccaat gacaagcttg ctagcggtgg gcaaccacaa ac 525071733DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 507ccgtttaaac aattctgcag gaatctagtt attaatagta atcaattacg gggtcattag 60ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 180caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 240cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 300ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 360tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc 420tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt 480gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg 540aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc 600gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc 660ggcgggcgga agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc 720gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc 780cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc 840tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg 900gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct 960gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg 1020cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc 1080ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc 1140ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta 1200cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg 1260gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggcccccgga 1320gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg 1380agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc 1440gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg 1500gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg 1560gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct 1620ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc 1680tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaa 17335084269DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 508atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat 60gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc 120gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 180accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 240agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 300acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 360ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 420gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 480atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 540ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 600atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 660gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 720aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 780ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 840attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 900gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 960atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 1020ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 1080atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1140cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1200tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1260aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1320cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1380attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1440aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1500ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1560gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1620ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1680aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1740ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1800aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1860ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1920aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1980accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 2040ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 2100ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2160ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2220ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2280gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2340aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2400gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2460aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2520gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2580atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2640gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2700aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2760tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2820aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2880gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2940aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 3000ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 3060caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3120cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3180atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3240atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3300ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3360accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3420acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3480agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3540tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3600gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3660ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3720tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3780aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3840tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3900cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3960ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 4020atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 4080gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4140gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4200ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 4260aagaaaaag 4269509780DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 509ggaagcggag ccactaactt ctccctgttg aaacaagcag gggatgtcga agagaatccc 60gggccagtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 120gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 180tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc

240accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 300aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 360ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 420ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 480cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 540aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 600gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 660cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg 720gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 780510597DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 510cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 60tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 120ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga 180gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc 240cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct 300ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg 360gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct 420gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc 480cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg 540tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcg 597511210DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 511cgacctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 60ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 120cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 180gggaggattg ggaagacaat ggcaggcatg 210512906DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 512ataacttcgt ataatgtatg ctatacgaag ttattcgcga tgaataaatg aaagcttgca 60gatctgcgac tctagaggat ctgcgactct agaggatcat aatcagccnt accacatttt 120gtagaggttt tactngcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa 180atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc 240aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 300tccaaactca tcaatgtatc ttatcatgtc tggatctgcg actctagagg atcataatca 360gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga 420acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg 480gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 540ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgcgactcta 600gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc 660acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat 720tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 780tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 840gatccccatc aagctgatcc ggaaccctta atataacttc gtataatgta tgctatacga 900agttat 9065131079DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 513caggccctcc gagcgtggtg gagccgttct gtgagacagc cgggtacgag tcgtgacgct 60ggaaggggca agcgggtggt gggcaggaat gcggtccgcc ctgcagcaac cggaggggga 120gggagaaggg agcggaaaag tctccaccgg acgcggccat ggctcggggg ggggggggca 180gcggaggagc gcttccggcc gacgtctcgt cgctgattgg cttcttttcc tcccgccgtg 240tgtgaaaaca caaatggcgt gttttggttg gcgtaaggcg cctgtcagtt aacggcagcc 300ggagtgcgca gccgccggca gcctcgctct gcccactggg tggggcggga ggtaggtggg 360gtgaggcgag ctggacgtgc gggcgcggtc ggcctctggc ggggcggggg aggggaggga 420gggtcagcga aagtagctcg cgcgcgagcg gccgcccacc ctccccttcc tctgggggag 480tcgttttacc cgccgccggc cgggcctcgt cgtctgattg gctctcgggg cccagaaaac 540tggcccttgc cattggctcg tgttcgtgca agttgagtcc atccgccggc cagcgggggc 600ggcgaggagg cgctcccagg ttccggccct cccctcggcc ccgcgccgca gagtctggcc 660gcgcgcccct gcgcaacgtg gcaggaagcg cgcgctgggg gcggggacgg gcagtagggc 720tgagcggctg cggggcgggt gcaagcacgt ttccgacttg agttgcctca agaggggcgt 780gctgagccag acctccatcg cgcactccgg ggagtggagg gaaggagcga gggctcagtt 840gggctgtttt ggaggcagga agcacttgct ctcccaaagt cgctctgagt tgttatcagt 900aagggagctg cagtggagta ggcggggaga aggccgcacc cttctccgga ggggggaggg 960gagtgttgca atacctttct gggagttctc tgctgcctcc tggcttctga ggaccgccct 1020gggcctggga gaatcccttc cccctcttcc ctcgtgatct gcaactccag tctttctag 10795144336DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 514agatgggcgg gagtcttctg ggcaggctta aaggctaacc tggtgtgtgg gcgttgtcct 60gcaggggaat tgaacaggtg taaaattgga gggacaagac ttcccacaga ttttcggttt 120tgtcgggaag ttttttaata ggggcaaata aggaaaatgg gaggataggt agtcatctgg 180ggttttatgc agcaaaacta caggttatta ttgcttgtga tccgcctcgg agtattttcc 240atcgaggtag attaaagaca tgctcacccg agttttatac tctcctgctt gagatcctta 300ctacagtatg aaattacagt gtcgcgagtt agactatgta agcagaattt taatcatttt 360taaagagccc agtacttcat atccatttct cccgctcctt ctgcagcctt atcaaaaggt 420attttagaac actcatttta gccccatttt catttattat actggcttat ccaaccccta 480gacagagcat tggcattttc cctttcctga tcttagaagt ctgatgactc atgaaaccag 540acagattagt tacatacacc acaaatcgag gctgtagctg gggcctcaac actgcagttc 600ttttataact ccttagtaca ctttttgttg atcctttgcc ttgatcctta attttcagtg 660tctatcacct ctcccgtcag gtggtgttcc acatttgggc ctattctcag tccagggagt 720tttacaacaa tagatgtatt gagaatccaa cctaaagctt aactttccac tcccatgaat 780gcctctctcc tttttctcca tttataaact gagctattaa ccattaatgg tttccaggtg 840gatgtctcct cccccaatat tacctgatgt atcttacata ttgccaggct gatattttaa 900gacattaaaa ggtatatttc attattgagc cacatggtat tgattactgc ttactaaaat 960tttgtcattg tacacatctg taaaaggtgg ttccttttgg aatgcaaagt tcaggtgttt 1020gttgtctttc ctgacctaag gtcttgtgag cttgtatttt ttctatttaa gcagtgcttt 1080ctcttggact ggcttgactc atggcattct acacgttatt gctggtctaa atgtgatttt 1140gccaagcttc ttcaggacct ataattttgc ttgacttgta gccaaacaca agtaaaatga 1200ttaagcaaca aatgtatttg tgaagcttgg tttttaggtt gttgtgttgt gtgtgcttgt 1260gctctataat aatactatcc aggggctgga gaggtggctc ggagttcaag agcacagact 1320gctcttccag aagtcctgag ttcaattccc agcaaccaca tggtggctca caaccatctg 1380taatgggatc tgatgccctc ttctggtgtg tctgaagacc acaagtgtat tcacattaaa 1440taaataaatc ctccttcttc ttcttttttt tttttttaaa gagaatactg tctccagtag 1500aatttactga agtaatgaaa tactttgtgt ttgttccaat atggtagcca ataatcaaat 1560tactctttaa gcactggaaa tgttaccaag gaactaattt ttatttgaag tgtaactgtg 1620gacagaggag ccataactgc agacttgtgg gatacagaag accaatgcag actttaatgt 1680cttttctctt acactaagca ataaagaaat aaaaattgaa cttctagtat cctatttgtt 1740taaactgcta gctttactta acttttgtgc ttcatctata caaagctgaa agctaagtct 1800gcagccatta ctaaacatga aagcaagtaa tgataatttt ggatttcaaa aatgtagggc 1860cagagtttag ccagccagtg gtggtgcttg cctttatgcc tttaatccca gcactctgga 1920ggcagagaca ggcagatctc tgagtttgag cccagcctgg tctacacatc aagttctatc 1980taggatagcc aggaatacac acagaaaccc tgttggggag gggggctctg agatttcata 2040aaattataat tgaagcattc cctaatgagc cactatggat gtggctaaat ccgtctacct 2100ttctgatgag atttgggtat tattttttct gtctctgctg ttggttgggt cttttgacac 2160tgtgggcttt ctttaaagcc tccttcctgc catgtggtct cttgtttgct actaacttcc 2220catggcttaa atggcatggc tttttgcctt ctaagggcag ctgctgagat ttgcagcctg 2280atttccaggg tggggttggg aaatctttca aacactaaaa ttgtccttta attttttttt 2340taaaaaatgg gttatataat aaacctcata aaatagttat gaggagtgag gtggactaat 2400attaaatgag tccctcccct ataaaagagc tattaaggct ttttgtctta tacttaactt 2460tttttttaaa tgtggtatct ttagaaccaa gggtcttaga gttttagtat acagaaactg 2520ttgcatcgct taatcagatt ttctagtttc aaatccagag aatccaaatt cttcacagcc 2580aaagtcaaat taagaatttc tgacttttaa tgttaatttg cttactgtga atataaaaat 2640gatagctttt cctgaggcag ggtctcacta tgtatctctg cctgatctgc aacaagatat 2700gtagactaaa gttctgcctg cttttgtctc ctgaatacta aggttaaaat gtagtaatac 2760ttttggaact tgcaggtcag attcttttat aggggacaca ctaagggagc ttgggtgata 2820gttggtaaat gtgtttaagt gatgaaaact tgaattatta tcaccgcaac ctacttttta 2880aaaaaaaaag ccaggcctgt tagagcatgc ttaagggatc cctaggactt gctgagcaca 2940caagagtagt tacttggcag gctcctggtg agagcatatt tcaaaaaaca aggcagacaa 3000ccaagaaact acagttaagg ttacctgtct ttaaaccatc tgcatataca cagggatatt 3060aaaatattcc aaataatatt tcattcaagt tttcccccat caaattggga catggatttc 3120tccggtgaat aggcagagtt ggaaactaaa caaatgttgg ttttgtgatt tgtgaaattg 3180ttttcaagtg atagttaaag cccatgagat acagaacaaa gctgctattt cgaggtctct 3240tggtttatac tcagaagcac ttctttgggt ttccctgcac tatcctgatc atgtgctagg 3300cctaccttag gctgattgtt gttcaaataa acttaagttt cctgtcaggt gatgtcatat 3360gatttcatat atcaaggcaa aacatgttat atatgttaaa catttgtact taatgtgaaa 3420gttaggtctt tgtgggtttg atttttaatt ttcaaaacct gagctaaata agtcattttt 3480acatgtctta catttggtgg aattgtataa ttgtggtttg caggcaagac tctctgacct 3540agtaacccta cctatagagc actttgctgg gtcacaagtc taggagtcaa gcatttcacc 3600ttgaagttga gacgttttgt tagtgtatac tagtttatat gttggaggac atgtttatcc 3660agaagatatt caggactatt tttgactggg ctaaggaatt gattctgatt agcactgtta 3720gtgagcattg agtggccttt aggcttgaat tggagtcact tgtatatctc aaataatgct 3780ggcctttttt aaaagccctt gttctttatc accctgtttt ctacataatt tttgttcaaa 3840gaaatacttg tttggatctc cttttgacaa caatagcatg ttttcaagcc atattttttt 3900tccttttttt tttttttttt ggtttttcga gacagggttt ctctgtatag ccctggctgt 3960cctggaactc actttgtaga ccaggctggc ctcgaactca gaaatccgcc tgcctctgcc 4020tcctgagtgc cgggattaaa ggcgtgcacc accacgcctg gctaagttgg atattttgtt 4080atataactat aaccaatact aactccactg ggtggatttt taattcagtc agtagtctta 4140agtggtcttt attggccctt cattaaaatc tactgttcac tctaacagag gctgttggta 4200ctagtggcac ttaagcaact tcctacggat atactagcag attaagggtc agggatagaa 4260actagtctag cgttttgtat acctaccagc tttatactac cttgttctga tagaaatatt 4320tcaggacatc tagctt 43365151846DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 515aattctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag 60ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca 120ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc 180ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg 240aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg 300taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc 360tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc 420gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt 480tctcctcttc ctcatctccg ggcctttcga cctgcaatcg ccgctagcga agttcctatt 540ctctagaaag tataggaact tcgccaccat gggatcggcc attgaacaag atggattgca 600cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 660aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 720tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 780gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 840aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 900tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 960ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 1020ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 1080cgaactgttc gccaggctca aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca 1140tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 1200ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 1260tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 1320tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaggggatcc 1380gctgtaagtc tgcagaaatt gatgatctat taaacaataa agatgtccac taaaatggaa 1440gtttttcctg tcatactttg ttaagaaggg tgagaacaga gtacctacat tttgaatgga 1500aggattggag ctacgggggt gggggtgggg tgggattaga taaatgcctg ctctttactg 1560aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcata atttaaacaa 1620gcaaaaccaa attaagggcc agctcattcc tcccactcat gatctataga tctatagatc 1680tctcgtggga tcattgtttt tctcttgatt cccactttgt ggttctaagt actgtggttt 1740ccaaatgtgt cagtttcata gcctgaagaa cgagatcagc agcctctgtt ccacatacac 1800ttcattctca gtattgtttt gccaagttct aattccatca gaaagc 18465161519DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 516taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg 60ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt 120aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta 180gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta 240gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc 300ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga 360aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg 420tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc 480tcttcctcat ctccgggcct ttcgacctgc aggtcctcgc catggatcct gatgatgttg 540ttgattcttc taaatctttt gtgatggaaa acttttcttc gtaccacggg actaaacctg 600gttatgtaga ttccattcaa aaaggtatac aaaagccaaa atctggtaca caaggaaatt 660atgacgatga ttggaaaggg ttttatagta ccgacaataa atacgacgct gcgggatact 720ctgtagataa tgaaaacccg ctctctggaa aagctggagg cgtggtcaaa gtgacgtatc 780caggactgac gaaggttctc gcactaaaag tggataatgc cgaaactatt aagaaagagt 840taggtttaag tctcactgaa ccgttgatgg agcaagtcgg aacggaagag tttatcaaaa 900ggttcggtga tggtgcttcg cgtgtagtgc tcagccttcc cttcgctgag gggagttcta 960gcgttgaata tattaataac tgggaacagg cgaaagcgtt aagcgtagaa cttgagatta 1020attttgaaac ccgtggaaaa cgtggccaag atgcgatgta tgagtatatg gctcaagcct 1080gtgcaggaaa tcgtgtcagg cgatctcttt gtgaaggaac cttacttctg tggtgtgaca 1140taattggaca aactacctac agagatttaa agctctaagg taaatataaa atttttaagt 1200gtataatgtg ttaaactact gattctaatt gtttgtgtat tttagattcc aacctatgga 1260actgatgaat gggagcagtg gtggaatgca gatcctagag ctcgctgatc agcctcgact 1320gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 1380gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 1440agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 1500gaagacaata gcaggcatg 151951723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 517gagggcctat ttcccatgat tcc 2351822DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 518cttgtggaaa ggacgaaaca cc 2251945DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 519aacnnnnnnn nnnnnnnnnn nnnggtgttt cgtcctttcc acaag 4552028DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 520cctgagtgtt gaggccccag tggctgct 2852137DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 521acgagggcag agtgctgctt gctgctggcc aggcccc 3752268DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 522catcaggctc tcagctcagc ctgagtgttg aggccctgct ggccaggccc ctgcgtgggc 60ccaagctg 6852327DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 523gagggcctat ttcccatgat tccttca 27524125DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 524aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa 60cttgctattt ctagctctaa aacnnnnnnn nnnnnnnnnn nnnggtgttt cgtcctttcc 120acaag 125525111DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 525gaaacaccnn nnnnnnnnnn nnnnnnnngt tttagagcta gaaatagcaa gttaaaataa 60ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt t 11152620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 526agctgtttta ctggtcggct 2052720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 527aatggataca cctggtcgaa 2052820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 528caatggatac acctggtcga 2052968DNAHomo sapiens 529accatgtata ccacttgggc tttggcagta gctaactgca ctaaatataa tataaggagg 60gttttatg 6853023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 530nnnnnnnnnn nnnnnnnnnn ngg 2353115DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 531nnnnnnnnnn nnngg 1553223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 532nnnnnnnnnn nnnnnnnnnn ngg 2353314DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 533nnnnnnnnnn nngg 1453425DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 534nnnnnnnnnn nnnnnnnnnn nggng 2553517DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 535nnnnnnnnnn nnnggng 1753625DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 536nnnnnnnnnn nnnnnnnnnn nggng

2553716DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 537nnnnnnnnnn nnggng 16

Claims

1. A method of inducing cell death of one or more prokaryotic cell(s) 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, and a tracr sequence; and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, are produced in the prokaryotic cell(s); wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within a 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 results in Cas9-directed cleavage at a targeted site in the prokaryote(s) and induces cell death.

2. The method of claim 1, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

3. The method of claim 1, wherein the CRISPR enzyme is a Cas9.

4. The method of claim 3, wherein the Cas9 is S. pyogenes Cas9.

5. The method of claim 1, wherein the CRISPR enzyme is not endogenous to the prokaryote(s).

6. The method of claim 1 wherein the prokaryote(s) is S. pneumoniae or E. coli.

7. The method of claim 1 wherein the one or more vectors are plasmids.