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CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation … — The Broad Institute, Inc. (US20200354742A1)

The Broad Institute, Inc. · Google Patents

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fengzhang thebroadinstitute

patent, google patents, intellectual property, US20200354742A1, The Broad Institute, Inc., Feng Zhang, en, 2020

ABSTRACT

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

This application is a continuation of U.S. application Ser. No. 15/229,702, filed on Aug. 5, 2016, which is a continuation of U.S. application Ser. No. 14/105,031, filed Dec. 12, 2013, which claims priority to U.S. provisional patent applications 61/736,527, 61/748,427, 61/791,409 and 61/835,931 all entitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Dec. 12, 2012, Jan. 2, 2013, Mar. 15, 2013 and Jun. 17, 2013, respectively. This application also claims priority to U.S. provisional patent application 61/802,174 entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Mar. 15, 2013.

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 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, entitled CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS filed on Jul. 2, 2013 and Oct. 15, 2013 respectively.

The foregoing applications, and all documents cited therein or during their prosecution (âappin cited documentsâ) and all documents cited or referenced in the appin cited documents, and all documents cited or referenced herein (âherein cited documentsâ), 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.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. MH100706 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

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 Aug. 28, 2014, is named 44790.06.2003_SL.txt and is 321,104 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that may use vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof.

BACKGROUND OF THE INVENTION

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

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.

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%, 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 âvectorâ 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 âplasmid,â 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 âexpression vectors.â Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

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, âoperably linkedâ 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).

The term âregulatory elementâ is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the Î²-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1Î± promoter. Also encompassed by the term âregulatory elementâ are enhancer elements, such as WPRE; CMV enhancers; the R-U5â² 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 Î²-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.).

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

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.

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.

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RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. MH100706 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

SUMMARY OF THE INVENTION

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

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.

In one aspect, the invention provides a kit comprising one or more of the components described herein. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence. In some embodiments, the kit comprises components (a) and (b) located on the same or different vectors of the system. In some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system further comprises a third regulatory element, such as a polymerase III promoter, operably linked to said tracr sequence. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length.

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.

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.

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.

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.

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.

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.

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. Â§ 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.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as âcomprisesâ, âcomprisedâ, âcomprisingâ and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean âincludesâ, âincludedâ, âincludingâ, and the like; and that terms such as âconsisting essentially ofâ and âconsists essentially ofâ 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

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:

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 5â²-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB) Ë3 bp upstream of the PAM (red triangle).

FIGS. 2A-2F show an exemplary CRISPR system, a possible mechanism of action, an example adaptation for expression in eukaryotic cells, and results of tests assessing nuclear localization and CRISPR activity. 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. FIG. 2B illustrates engineering of S. pyogenes Cas9 (SpCas9) and RNase III (SpRNase III) with nuclear localization signals (NLSs) to enable import into the mammalian nucleus. FIG. 2C illustrates mammalian expression of SpCas9 and SpRNase III driven by the constitutive EF1a promoter and tracrRNA and pre-crRNA array (DR-Spacer-DR) driven by the RNA Pol3 promoter U6 to promote precise transcription initiation and termination. 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. FIG. 2C discloses SEQ ID NOS 281-282, respectively, in order of appearance. FIG. 2E discloses SEQ ID NOS 283-285, respectively, in order of appearance. FIG. 2F discloses SEQ ID NOS 286-290, respectively, in order of appearance.

FIG. 3A-3C shows an exemplary expression cassette for expression of CRISPR system elements in eukaryotic cells, predicted structures of example guide sequences, and CRISPR system activity as measured in eukaryotic and prokaryotic cells (SEQ ID NOS 291-300, respectively, in order of appearance). 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. FIG. 3B provides a schematic showing guide sequences targeting the human EMX1, PVALB, and mouse Th loci, as well as their predicted secondary structures. FIG. 3C shows evaluation of crRNA targeting efficiency.

FIGS. 4A-4D show results of an evaluation of SpCas9 specificity for an example target. FIG. 4A shows analysis of the effect of single-nucleotide mutations in the guide sequence on protospacer cleavage in the mammalian genome using a series of EMX1-targeting chimeric crRNAs with single point mutations. FIG. 4A discloses SEQ ID NOS 301, 284 and 302-312, respectively, in order of appearance. FIG. 4B illustrates results of a Surveyor nuclease assay comparing the cleavage efficiency of Cas9 when paired with different mutant chimeric RNAs. FIG. 4C provides a schematic showing the design of TALENs targeting EMX1. FIG. 4C discloses SEQ ID NO: 301. FIG. 4D shows a Surveyor gel comparing the efficiency of TALEN and Cas9 (n=3).

FIGS. 5A-5G show an exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells. FIG. 5A discloses SpCas9n. FIG. 5B discloses co-expression of EMX1-targeting chimeric crRNA with SpCas9 produced indels in the target site, whereas co-expression with SpCas9n did not. FIG. 5C provides a schematic illustration of the HR strategy. FIG. 5D shows restriction fragment length polymorphism gel analysis. FIG. 5E discloses SEQ ID NO: 313. FIG. 5F discloses SEQ ID NOS 314-315, respectively, in order of appearance. FIG. 5G discloses SEQ ID NOS 316-320, respectively, in order of appearance.

FIG. 6 provides a table of protospacer sequences (

SEQ ID NOS

33, 32, 31, 324-329, 35, 34 and 332-336, 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).

FIGS. 7A-7C show a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting. FIG. 7A discloses SEQ ID NOS 337-338, respectively, in order of appearance. FIG. 7A shows expression constructs for tracrRNA. FIG. 7B shows results of the Surveyor assay. 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.

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

FIGS. 9A-9B show exemplary bicistronic expression vectors for expression of CRISPR system elements in eukaryotic cells. FIG. 9A discloses SEQ ID NOS 339-341, respectively, in order of appearance. FIG. 9B discloses SEQ ID NOS 342-344, respectively, in order of appearance.

FIG. 10A-10D shows a bacterial plasmid transformation interference assay, expression cassettes and plasmids used therein, and transformation efficiencies of cells used therein. FIG. 10A shows pCRISPR plasmid comprising elements of the S. pyogenes

CRISPR locus 1 sufficient for CRISPR activity in E. coli . FIG. 10A discloses SEQ ID NOS 345-347, respectively, in order of appearance. FIG. 10B shows challenge plasmids used in the interference assay were constructed by inserting the protospacer sequence along with an adjacent CRISPR motif sequence (PAM) into pUC19. FIG. 10C provides a schematic representation of the interference assay. FIG. 10D illustrates competence of each pCRISPR-carrying E. coli strain used in assays illustrated in FIG. 4C .

FIGS. 11A-11C show 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. 11C ).

FIGS. 12A-12C show an exemplary CRISPR system, an example adaptation for expression in eukaryotic cells, and results of tests assessing CRISPR activity. 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. FIG. 12B discloses SEQ ID NOS 348-349, respectively, in order of appearance. FIG. 12C provides a schematic showing guide sequences targeting the human EMX1 locus as well as their predicted secondary structures. FIG. 12C discloses SEQ ID NO: 350.

FIGS. 13A-13C show exemplary manipulations of a CRISPR system for targeting of 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. 13A discloses SEQ ID NO: 351. 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). FIG. 13B discloses SEQ ID NOS 352-354, respectively, in order of appearance. FIG. 13C shows results of a Surveyor assay comparing the efficacy of Cas9-mediated cleavage at five protospacers in the human EMX1 locus.

FIGS. 14A-14B show the results of a 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. FIG. 14A discloses SEQ ID NO: 355. FIG. 14B shows a Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying DR-EMX1(1)-DR.

FIG. 15A-15B shows an exemplary selection of protospacers in the human PVALB and mouse Th loci. FIG. 15A discloses SEQ ID NO: 356. FIG. 15B discloses SEQ ID NO: 357.

FIG. 16 shows example protospacer and corresponding PAM sequence targets of the S. thermophilus CRISPR system in the human EMX1 locus (SEQ ID NO: 350).

FIG. 17 provides a table of sequences for primers and probes (SEQ ID NOS 36-39 and 358-365, respectively, in order of appearance) used for Surveyor, RFLP, genomic sequencing, and Northern blot assays.

FIGS. 18A-18C show exemplary manipulation of a CRISPR system with chimeric RNAs and results of SURVEYOR assays for system activity in eukaryotic cells. FIG. 18A illustrates a schematic of a bicistronic expression vector for chimeric RNA and Cas9. FIG. 18A discloses SEQ ID NO: 366, respectively, in order of appearance. FIGS. 18B and 18C show results of SURVEYOR assays for Cas9-mediated indels at the human EMX1 and PVALB loci, respectively.

FIGS. 19A-19B show a graphical representation of the results of SURVEYOR assays for CRISPR system activity in eukaryotic cells. FIG. 19A shows results from the EMX1 locus. FIG. 19B shows results from the PVALB locus.

FIG. 20 shows an exemplary visualization of some S. pyogenes Cas9 target sites in the human genome using the UCSC genome browser (SEQ ID NOS 367-445, respectively, in order of appearance).

FIG. 21 shows predicted secondary structures for exemplary chimeric RNAs comprising a guide sequence, tracr mate sequence, and tracr sequence (SEQ ID NOS 446-465, respectively, in order of appearance).

FIG. 22 shows exemplary bicistronic expression vectors for expression of CRISPR system elements in eukaryotic cells ( SEQ ID NOS 466 and 343-344, respectively, in order of appearance).

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 8232.5 cells with no editing template, the R6 wild-type srtA or the R6370.1 editing templates. Recombination of either R6 srtA or R6 370.1 prevented cleavage by Cas9. Transformation efficiency was calculated as colony forming units (cfu) per Î¼g 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. âUn.â indicates the unedited srtA locus of strain R6 8232.5 ; âEd.â shows the editing template. R6 8232.5 and R6 370.1 targets are distinguished by restriction with EaeI.

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 467-471, respectively, in order of appearance). These cells expressed Cas9 loaded with a crRNA that targeted a chromosomal region of R6 8232.5 cells (highlighted in pink) that is absent from the R6 genome. More than 2Ã105 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: 472). 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 âAnalysis of deep sequencing dataâ in Example 5)

FIG. 25A-25F shows introduction of single and multiple mutations using the CRISPR system in S. pneumoniae . ( FIG. 25A ) 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 473-477 and 474, respectively, in order of appearance). ( FIG. 25B ) Transformation efficiency of cells transformed with targeting constructs in the presence of an editing template or control. ( FIG. 25C ) PCR analysis for 8 transformants of each editing experiment followed by digestion with BtgZI (RâA) and TseI (NEâAA). Deletion of bgaA was revealed as a smaller PCR product. ( FIG. 25D ) Miller assay to measure the Î²-galactosidase activity of WT and edited strains. ( FIG. 25E ) 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 ( FIG. 25F ) PCR analysis for 8 transformants to detect deletions in srtA and bgaA loci. 6/8 transformants contained deletions of both genes.

FIG. 26A-26D provides mechanisms underlying editing using the CRISPR system. ( FIG. 26A ) 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. ( FIG. 26B ) Mutant and wild-type ermAM sequences (SEQ ID NOS 478-481, respectively, in order of appearance). ( FIG. 26C ) Fraction of erythromicyn-resistant (erm R ) cfu calculated from total or kanamycin-resistant cfu. ( FIG. 26D ) 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 meanÂ±s.d. for three independent experiments.

FIG. 27A-27D illustrates genome editing with the CRISPR system in E. coli . ( FIG. 27A ) A kanamycin-resistant plasmid carrying the CRISPR array (pCRISPR) targeting the gene to edit may be transformed in the H1VIE63 recombineering strain containing a chloramphenicol-resistant plasmid ha

CLAIMS

Claims ( 59 )

1 . An engineered composition comprising a vector system comprising one or more eukaryotic expression vectors comprising

I. a nucleotide sequence encoding a CRISPR-Cas complex chimeric RNA (chiRNA) having two or more hairpins and comprising (a) a guide sequence capable of hybridizing to a target eukaryotic genome DNA sequence adjacent to a Protospacer Adjacent Motif (PAM) in a eukaryotic cell (b) a tracr mate sequence, and (c) a tracr sequence comprising 30 or more nucleotides in length, and II. a nucleotide sequence encoding a Cas9 comprising one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9 and one or more mutations in a catalytic domain thereby rendering it a nickase that cleaves a single DNA strand, wherein:

components I and II are located on the same or different vectors of the system, and

components I and II each are operably linked to a regulatory element for transcription thereof in the eukaryotic cell and there can be one or more regulatory elements for transcription of components I and II.

2 . A CRISPR composition for multiplexing, wherein the composition comprises a vector system comprising one or more eukaryotic expression vectors comprising

I. a nucleotide sequence encoding more than one CRISPR-Cas complex each having two or more hairpins and comprising

(a) a guide sequence capable of hybridizing to a different target eukaryotic genome DNA sequence adjacent to a PAM in a eukaryotic,

(b) a tracr mate sequence, and

II. a nucleotide sequence encoding a Cas9 comprising one or more NLSs in the proximity of a terminus of the Cas9 and one or more mutations in a catalytic domain thereby rendering it a nickase that cleaves a single DNA strand,

wherein:

each of components I and II are operably linked to a regulatory element for transcription thereof in the eukaryotic cell and there can be one or more regulatory elements for transcription of components I and II.

3 . The composition of claim 1 , wherein the regulatory element operably linked to component I comprises a polymerase III promoter.

4 . The composition of claim 1 , wherein the regulatory element operably linked to component II comprises a polymerase II promoter.

5 . The composition of claim 1 , wherein the guide sequence comprises about or more than about 10-75 nucleotides in length, and the tracr sequence comprises about or more than about 30-50 nucleotides in length, and the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate.

6 . The composition of claim 1 , wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence.

7 . The composition of claim 1 , wherein the nucleotide sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell.

8 . The composition of claim 1 , wherein the guide sequence is at least 15 nucleotides in length.

9 . The composition of claim 1 , wherein the chiRNA comprises two, three, four or five hairpins.

10 . The composition of claim 1 , wherein the catalytic domain comprises RuvCI, RuvCII, RuvCIII or HNH domain.

11 . The composition of claim 1 , wherein the Cas9 comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986, with reference to S. pyogenes Cas9.

12 . The composition of claim 1 , wherein the Cas9 comprises a mutation comprising D10A, E762A, H840A, N854A, N863A or D986A, with reference to S. pyogenes Cas9.

13 . An engineered composition comprising a vector system comprising one or more vectors comprising

I. a nucleotide sequence encoding (a) a guide sequence capable of hybridizing to a eukaryotic genome DNA target sequence adjacent to a PAM in a eukaryotic cell, and (b) a tracr mate sequence, II. a nucleotide sequence encoding a Cas9 comprising one or more NLSs in the proximity of a terminus of the Cas9 and one or more mutations in a catalytic domain thereby rendering it a nickase that cleaves a single DNA strand, and III. a nucleotide sequence encoding a tracr sequence comprising 30 or more nucleotides in length, wherein:

components I, II and III are located on the same or different vectors of the system, and

components I, II and III are each operably linked to a regulatory element and there can be one or more regulatory elements for transcription of components I, II and III.

14 . A CRISPR composition for multiplexing, wherein the composition comprises a vector system comprising one or more vectors comprising

I. (a) nucleotide sequences encoding more than one guide sequences each capable of hybridizing to a different eukaryotic genome DNA target sequence adjacent to a PAM in a eukaryotic cell, and (b) a tracr mate sequence, II. a nucleotide sequence encoding a Cas9 comprising one or more NLSs in the proximity of a terminus of the Cas9 and one or more mutations in a catalytic domain thereby rendering it a nickase that cleaves a single DNA strand, and III. a nucleotide sequence encoding a tracr sequence comprising 30 or more nucleotides in length, wherein:

components I, II and III are located on the same or different vectors of the system, and

each of components I, II and III are operably linked to a regulatory element for transcription thereof in the eukaryotic cell and there can be one or more regulatory elements therefor.

15 . The composition of claim 13 , wherein the regulatory element operably linked to component I comprises a polymerase III promoter.

16 . The composition of claim 13 , wherein the regulatory element operably linked to component II comprises a polymerase II promoter.

17 . The composition of claim 13 , wherein the regulatory element operably linked to component III comprises a polymerase III promoter.

18 . The composition of claim 13 , wherein the guide sequence comprises about or more than about 10-75 nucleotides in length, and the tracr sequence comprises about or more than about 30-50 nucleotides in length, and the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate.

19 . The composition of claim 13 , wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence.

20 . The composition of claim 13 , wherein the nucleotide sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell.

21 . The composition of claim 13 , wherein the guide sequence is at least 15 nucleotides in length.

22 . The composition of claim 13 , wherein the catalytic domain comprises RuvCI, RuvCII, RuvCIII or HNH domain.

23 . The composition of claim 13 , wherein the Cas9 comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986, with reference to S. pyogenes Cas9.

24 . The composition of claim 13 , wherein the Cas9 comprises a mutation comprising D10A, E762A, H840A, N854A, N863A or D986A, with reference to S. pyogenes Cas9.

25 . An isolated eukaryotic host cell comprising the composition of claim 1 .

26 . A non-human animal comprising the eukaryotic host cell of claim 25 .

27 . A non-human organism or plant comprising the eukaryotic host cell of claim 25 .

28 . A kit comprising the composition of claim 1 , and instructions for using said kit.

29 . A method of altering the expression of a genomic locus of interest in a eukaryotic cell comprising

contacting the eukaryotic cell with the composition of claim 1 and thereby delivering the one or more vectors and allowing a CRISPR-Cas complex to form and bind to target and determining if the expression of the genomic locus has been altered.

30 . The composition of claim 1 , wherein components I and II are on the same vector.

31 . An isolated eukaryotic cell or a eukaryotic cell of a non-human organism comprising the composition of claim 30 .

32 . The composition of claim 13 , wherein components I, II and III are on the same vector.

33 . An isolated eukaryotic cell or a eukaryotic cell of a non-human organism comprising the composition of claim 32 .

34 . The composition of claim 1 , wherein the Cas9 comprises one or more heterologous effector domains.

35 . The composition according to claim 34 , wherein the one or more effector domains comprises a transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, transcription-protein recruiting domain, cellular uptake activity associated domain, nucleic acid binding domain or antibody presentation domain.

36 . A method of modifying the target eukaryotic genome DNA sequence adjacent to a PAM in the eukaryotic cell, the method comprising contacting the eukaryotic cell with the composition of claim 1 and thereby delivering the one or more vectors to the eukaryotic cell and allowing a CRISPR complex to form and bind to the target to effect DNA cleavage of said target thereby modifying the target.

37 . The method of claim 36 , wherein said one or more vectors are delivered to the eukaryotic cell in a subject.

38 . The method of claim 36 , wherein said modifying takes place in said eukaryotic cell in a cell culture.

39 . The method of claim 36 , further comprising isolating said eukaryotic cell from a subject prior to said modifying.

40 . The method of claim 39 , further comprising returning said eukaryotic cell and/or progeny cell(s) therefrom to said subject after said modifying.

41 . An isolated eukaryotic cell or a eukaryotic cell of a non-human organism from the method of claim 36 .

42 . A vector comprising a regulatory element operably linked to a coding sequence encoding a Cas9 comprising one or more NLSs, wherein said regulatory element is configured to drive transcription of the Cas9 in a eukaryotic cell in an amount effective to accumulate the Cas9 in the nucleus of the eukaryotic cell; wherein the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 a nickase that cleaves a single DNA strand.

43 . The vector of claim 42 , wherein said regulatory element is a polymerase II promoter.

44 . An isolated eukaryotic cell or a eukaryotic cell of a non-human organism comprising the vector of claim 42 .

45 . An isolated eukaryotic cell or a eukaryotic cell of a non-human organism comprising a Cas9; wherein the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 a nickase that cleaves a single DNA strand.

46 . An isolated eukaryotic cell culture from the cell of claim 31 .

47 . An isolated eukaryotic cell culture from the cell of claim 33 .

48 . An isolated eukaryotic cell culture from the cell of claim 41 .

49 . An isolated eukaryotic cell culture from the cell of claim 44 .

50 . The composition of claim 1 wherein component I is operably linked to a first regulatory element and component II is operably linked to a second regulatory element.

51 . The composition of claim 13 wherein component I is operably linked to a first regulatory element, component II is operably linked to a second regulatory element, and component III is operably linked to a third regulatory element.

52 . The composition of claim 1 wherein the target eukaryotic genome DNA sequence adjacent to a PAM in a eukaryotic cell is a target mammalian genome DNA sequence adjacent to a PAM in a mammalian cell.

53 . A method of modifying the target mammalian genome DNA sequence adjacent to a PAM in the mammalian cell, the method comprising contacting the mammalian cell with the composition of claim 52 and thereby delivering the vector to the mammalian cell and allowing a CRISPR complex to form and bind to the target to effect DNA cleavage of said target, thereby modifying the target.

54 . The method of claim 53 wherein the modifying obtains increased expression as to a product of the mammalian cell, in comparison with a cell that has not been subjected to the method.

55 . The method of claim 36 wherein the modifying obtains increased expression as to a product of the cell, in comparison with a cell that has not been subjected to the method.

56 . An isolated cell having increased expression as to a product of the cell, from the method of claim 54 , wherein the increased expression is in comparison with a cell that has not been subjected to method modifying the target genome DNA sequence in the cell.

57 . An isolated cell having increased expression as to a product of the cell, from the method of claim 55 , wherein the increased expression is in comparison with a cell that has not been subjected to method modifying the target genome DNA sequence in the cell.

58 . An isolated cell line of the cell of claim 56 .

59 . An isolated cell line of the cell of claim 57 .

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