Systems, methods and compositions for sequence manipulation with optimized functional crispr-cas systems

Abstract

To provide systems, methods and compositions for altering expression of target gene sequences and related gene products.SOLUTION: Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system. In particular, the present invention comprehends optimized functional CRISPR-Cas enzyme systems.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications and Incorporation by Reference This application claims priority from U.S. Provisional Patent Application Nos. 61 / 915,251, filed December 12, 2013; 61 / 930,214, filed January 22, 2014; 61 / 939,242, filed February 12, 2014; 61 / 980,012, filed April 15, 2014; 62 / 055,484, filed September 25, 2014; 62 / 087,537, filed November 4, 2014; 61 / 915,267, filed December 12, 2013; and 61 / 939,256, filed February 12, 2014.

[0002] The above applications, and all documents cited in those applications or during their prosecution ("application cited documents"), and all documents cited or referenced in those application cited documents, as well as all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in the herein cited documents, together with any manufacturer's instructions, manuals, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of this invention. More specifically, all references are incorporated by reference to the same extent as if each individual document were individually and specifically indicated to be incorporated by reference.

[0003] The present invention generally relates to systems, methods and compositions used for controlling gene expression, including sequence targeting, for example, genome perturbation or gene editing, which can use vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its components.In particular, the present invention encompasses an optimized function CRISPR-Cas enzyme system.

[0004] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with government support under NIH Pioneer Award DP1MH100706 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] This invention was made with government support under the PRESTO (Strategic Basic Research Program, PRESTO) in the field of "Structural Life Science and Advanced Fundamental Technologies Aiming for Innovation in Life Sciences" funded by JST (Japan Science and Technology Agency) in 2012. JST has certain rights in this invention.

[0006] This invention was made with government support under the category of "Development of New CRISPR Cas9 System Set and Its Medical Application" awarded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in 2014. MEXT has certain rights in this invention. [Background technology]

[0007] Recent advances in genome sequencing technology and analytical methods have significantly accelerated the ability to classify and map genetic factors related to a wide range of biological functions and diseases.Accurate genome targeting technology is needed to enable the systematic reverse engineering of causal gene mutations by enabling the selective perturbation of individual genetic elements, and to advance synthetic biology, biotechnology and pharmaceutical applications.Genome editing technology, such as designer zinc finger, transcription activator-like effector (TALE), or homing meganuclease, can be used to produce targeted genome perturbations, but there is still a need for new genome engineering technology that is inexpensive, easy to set up, scalable, and easy to target multiple locations in eukaryotic genomes. Summary of the Invention [Means for solving the problem]

[0008] There is an urgent need for alternative, robust systems and technologies for versatile sequence targeting. The present invention addresses this need and provides related advantages. CRISPR / Cas or CRISPR-Cas systems (both terms are used interchangeably throughout this application) do not require the generation of customized proteins to target specific sequences, but rather allow a single Cas enzyme to 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 the short RNA molecule. The addition of CRISPR-Cas systems to the repertoire of genome sequencing technologies and analytical methods significantly simplifies methodology and accelerates the ability to classify and map genetic factors associated with a diverse range of biological functions and diseases. To effectively utilize CRISPR-Cas systems for genome editing without adverse effects, it is important to understand the engineering and optimization aspects of these genome engineering tools, which are an embodiment of the claimed invention.

[0009] In one embodiment, the present invention provides a non-naturally occurring or engineered composition comprising a guide RNA (sgRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein at least one loop of the sgRNA is modified with the insertion of a distinct RNA sequence that binds to one or more adaptor proteins, and the adaptor protein is associated with one or more functional domains. When two or more functional domains are present, the functional domains can be the same or different, for example, two identical or two different activators or repressors. In one embodiment, the present invention provides a non-naturally occurring or engineered CRISPR-Cas complex composition comprising an sgRNA and a CRISPR enzyme as described herein. In one embodiment, the present invention provides a non-naturally occurring or engineered CRISPR-Cas complex composition as described herein, wherein the CRISPR enzyme comprises at least one mutation such that the CRISPR enzyme has 5% or less of the nuclease activity of a CRISPR enzyme without the at least one mutation; and / or comprises at least one or more nuclear localization sequences.

[0010] In one aspect, the invention provides an sgRNA or CRISPR-Cas complex as described herein, wherein one or more adaptor proteins are associated with one or more functional domains and are bound to a distinct RNA sequence inserted into at least one loop of the sgRNA.

[0011] In one aspect, the invention provides a non-naturally occurring or engineered composition comprising a CRISPR enzyme, the composition comprising one or more guide RNAs (sgRNAs) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, the CRISPR enzyme comprising at least one or more nuclear localization sequences, the CRISPR enzyme comprising at least one mutation such that the CRISPR enzyme has 5% or less of the nuclease activity of a CRISPR enzyme without the at least one mutation, and at least one loop of at least one sgRNA is modified with an insertion of a distinct RNA sequence that binds to one or more adaptor proteins, the adaptor proteins being associated with one or more functional domains.

[0012] In one embodiment, the present invention provides any of the compositions described herein, wherein the CRISPR enzyme has at least 97%, or 100%, reduced nuclease activity compared to a CRISPR enzyme that does not have at least one mutation. In one embodiment, the present invention provides any of the above compositions, wherein the CRISPR enzyme comprises two or more mutations, including mutations at two or more of D10, E762, H840, N854, N863, or D986 in the SpCas9 protein or any corresponding orthologue, or N580 in the SaCas9 protein, or wherein the CRISPR enzyme comprises at least one mutation, including mutations at least at H840. In one embodiment, the present invention provides a composition described herein, wherein the CRISPR enzyme comprises two or more mutations, including D10A, E762A, H840A, N854A, N863A, or D986A in the SpCas9 protein or any corresponding orthologue, or N580A in the SaCas9 protein, or at least one mutation, including H840A. In one embodiment, the present invention provides any composition described herein, wherein the CRISPR enzyme comprises H840A, or D10A and H840A, or D10A and N863A, by an SpCas9 protein or any corresponding orthologue.In one embodiment, the present invention provides any composition described herein, wherein the CRISPR enzyme comprises N580A, by a SaCas9 protein or any corresponding orthologue; or D10A, by an SpCas9 protein or any corresponding orthologue, and N580A, by a SaCas9 protein.

[0013] In one embodiment, the present invention provides any composition described herein, wherein CRISPR enzyme is associated with one or more functional domains.In one embodiment, the present invention provides any composition described herein, wherein one or more functional domains associated with adaptor protein are heterologous functional domains.In one embodiment, the present invention provides any composition described herein, wherein one or more functional domains associated with CRISPR enzyme are heterologous functional domains.

[0014] In one aspect, the invention provides a composition described herein, wherein the adaptor protein is a fusion protein comprising a functional domain, the fusion protein optionally comprising a linker between the adaptor protein and the functional domain, the linker optionally comprising a GlySer linker.

[0015] In one aspect, the invention provides a composition described herein, or any one of the preceding claims, wherein said at least one loop of the sgRNA is unmodified with the insertion of a distinct RNA sequence that binds to one or more adaptor proteins, and optionally one of the unmodified sgRNA loops is one of a tetraloop or stem-loop 2.

[0016] In one aspect, the invention provides a composition described herein, wherein the one or more functional domains associated with the adaptor protein are transcription activation domains.

[0017] In one embodiment, the invention provides a composition described herein, wherein the one or more functional domains associated with the CRISPR enzyme are transcription activation domains.

[0018] In one embodiment, the present invention provides a composition described herein, wherein one or more functional domains associated with an adaptor protein are transcription activation domains, including VP64, p65, MyoD1, HSF1, RTA, or SET7 / 9. Other references herein to activation (or activator) domains in relation to those associated with adaptor proteins include any known transcription activation domain, specifically VP64, p65, MyoD1, HSF1, RTA, or SET7 / 9.

[0019] In one embodiment, the present invention provides compositions as described herein, wherein one or more functional domains associated with CRISPR enzyme are transcription activation domains, including VP64, p65, MyoD1, HSF1, RTA and SET7 / 9.Other references herein to the activation (or activator) domain associated with CRISPR enzyme include any known transcription activation domain, particularly VP64, p65, MyoD1, HSF1, RTA or SET7 / 9.

[0020] In one embodiment, the invention provides a composition described herein, wherein one or more functional domains associated with the adaptor protein is a transcriptional repressor domain.

[0021] In one embodiment, the invention provides a composition described herein, wherein one or more functional domains associated with the CRISPR enzyme is a transcriptional repressor domain.

[0022] In one aspect, the invention provides a composition described herein, wherein the transcriptional repressor domain is a KRAB domain.

[0023] In one aspect, the invention provides a composition described herein, wherein the transcriptional repressor domain is a NuE domain, an NcoR domain, a SID domain, or a SID4X domain.

[0024] In one aspect, the invention provides a composition described herein, wherein one or more functional domains associated with the adaptor protein have one or more activities including methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, or nucleic acid binding activity.

[0025] In one embodiment, the invention provides a composition described herein, wherein one or more functional domains associated with the CRISPR enzyme have one or more activities including methylase activity, demethylase activity, transcriptional activator activity, transcriptional repressor activity, transcriptional release factor activity, histone modifying activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, nucleic acid binding activity, or molecular switch activity, or chemically or light-inducible.

[0026] Histone modification domains are also preferred in some embodiments. Exemplary histone modification domains are described below. Transposase, HR (homologous recombination) machinery domain, recombinase domain, and / or integrase domain are also preferred as functional domains of the present invention. In some embodiments, DNA integration activity includes HR machinery domain, integrase domain, recombinase domain, and / or transposase domain.

[0027] In one aspect, the invention provides a composition described herein, wherein the DNA cleavage activity is attributable to a nuclease.

[0028] In one aspect, the invention provides a composition described herein, wherein the nuclease comprises a Fok1 nuclease.

[0029] In one aspect, the invention provides a composition described herein, wherein one or more functional domains are attached to the CRISPR enzyme such that, upon binding to the sgRNA and target, the functional domains are in a spatial orientation that allows the functional domains to function in their attributed function.

[0030] In one embodiment, the present invention provides a composition described herein, wherein one or more functional domains are attached to a CRISPR enzyme via a linker, optionally a GlySer linker. In one embodiment, the present invention provides a composition described herein, wherein the sgRNA is modified such that the functional domains are in a spatial orientation that allows them to function in their assigned function after the sgRNA binds to an adaptor protein and further binds to the CRISPR enzyme and target.

[0031] In one embodiment, the invention provides a composition described herein, wherein at least one loop of the sgRNA is a tetraloop and / or loop 2.

[0032] In one aspect, the invention provides a composition described herein, wherein the tetraloop and loop 2 of the sgRNA are modified with the insertion of a distinct RNA sequence.

[0033] In one embodiment, the invention provides a composition described herein, wherein the insert of a distinct RNA sequence that binds to one or more adaptor proteins is an aptamer sequence.

[0034] In one aspect, the present invention provides a composition described herein, wherein the aptamer sequences are two or more aptamer sequences specific for the same adaptor protein.

[0035] In one aspect, the present invention provides a composition described herein, wherein the aptamer sequences are two or more aptamer sequences specific for different adaptor proteins.

[0036] In one aspect, the invention provides a composition described herein, wherein the adaptor protein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, or PRR1.

[0037] In one aspect, the invention provides a composition described herein, wherein the cell is a eukaryotic cell, optionally a mammalian cell.

[0038] In one aspect, the invention provides a composition described herein, wherein the cell is a human cell or a mouse cell.

[0039] In one aspect, the invention provides a mammalian cell as described herein, including, for example, a cell line, optionally a human cell line or a murine cell line.

[0040] In one aspect, the invention provides a transgenic mammal model, optionally a mouse, transformed with, or progeny of, a composition described herein.

[0041] In one aspect, the present invention provides a method of introducing a genomic locus event comprising administering to a host, or expressing in the host, one or more of the compositions described herein.

[0042] In one aspect, the invention provides a method as described herein, wherein the genomic locus event comprises affecting gene activation, gene inhibition, or a cleavage or insertion of DNA in the locus.

[0043] In one aspect, the invention provides a method described herein, comprising delivery of the composition or a nucleic acid molecule encoding same, wherein said nucleic acid molecule is operably linked to a regulatory sequence and is expressed in vivo. In vivo expression can be via lentivirus, adenovirus, or AAV.

[0044] In one aspect, the invention provides a vector comprising a nucleic acid molecule encoding a guide RNA (sgRNA), the vector comprising a regulatory element operable in a eukaryotic cell comprising the guide sequence (sgRNA) operably linked to a promoter, wherein the sgRNA is capable of hybridizing to a target sequence in a genomic locus of interest in the cell, and at least one loop of the sgRNA is modified with the insertion of a distinct RNA sequence that binds to one or more adaptor proteins.

[0045] In one aspect, the invention provides a vector comprising a regulatory element operable in a eukaryotic cell comprising a nucleic acid molecule encoding a CRISPR enzyme operably linked to a promoter, wherein the enzyme comprises at least one or more nuclear localization sequences; the CRISPR enzyme comprises at least one mutation such that the CRISPR enzyme has 5% or less of the nuclease activity of a CRISPR enzyme without said at least one mutation; and the CRISPR enzyme is associated with one or more functional domains.

[0046] In one aspect, the invention provides a nucleic acid molecule encoding an sgRNA or a CRISPR-Cas complex or composition described herein.

[0047] In one aspect, the invention provides a method of screening for gain of function (GOF) or loss of function (LOF), comprising cells of a cell line or model described herein that contain or express Cas9, the method comprising introducing into the cells of the cell line or model a composition of claim 1 (wherein the sgRNA comprises either an activator or a repressor), and monitoring GOF or LOF, respectively, for those cells where the introduced sgRNA comprises an activator, or for those cells where the introduced sgRNA comprises a repressor.

[0048]

[0013] In one embodiment, a CRISPR complex is provided, comprising a CRISPR enzyme and a guide RNA (sgRNA), wherein the CRISPR enzyme comprises at least one mutation such that the CRISPR enzyme has 5% or less of the nuclease activity of a CRISPR enzyme without the at least one mutation, and optionally at least one or more nuclear localization sequences; the guide RNA (sgRNA) comprises a guide sequence that can hybridize to a target sequence in a genomic locus of interest in a cell; the CRISPR enzyme is associated with two or more functional domains, which may be the same or different, preferably different functional domains; or at least one loop of the sgRNA is modified with an insertion of a distinct RNA sequence that binds to one or more adaptor proteins, wherein the adaptor proteins are associated with two or more functional domains; or the CRISPR enzyme is associated with one or more functional domains, and at least one loop of the sgRNA is modified with an insertion of a distinct RNA sequence that binds to one or more adaptor proteins, wherein the adaptor proteins are associated with one or more functional domains.

[0049] In one embodiment, the present invention provides a composition as described herein, wherein the CRISPR enzyme comprises one or more functional domains.In such a composition, two or more sgRNAs can be present, and the sgRNAs target different sequences, thereby allowing multiplexing when using the composition.The composition can comprise two or more sgRNAs modified with the insertion of distinct RNA sequences that bind to one or more adaptor proteins.The composition can comprise one or more adaptor proteins that are associated with one or more functional domains and that bind to distinct RNA sequences inserted into at least one loop of the sgRNA.

[0050] In one aspect, the present invention provides a method for altering or modifying the expression of a gene product. The method may include introducing into a cell containing and expressing a DNA molecule encoding one or more gene products an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein and one or more guide RNAs targeting the DNA molecule, whereby the guide RNA targets the DNA molecule encoding the gene product and the Cas protein cleaves the DNA molecule encoding the gene product, thereby altering the expression of the gene product; the Cas protein and the guide RNA do not occur together in nature. The present invention encompasses a guide RNA comprising a guide sequence fused to a tracr sequence. The present invention further encompasses a Cas protein that is codon-optimized for expression in eukaryotic cells. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the present invention, the expression of a gene product is reduced.

[0051] In one embodiment, in the compositions described herein, the target sequence can be a non-coding or regulatory (eg, a promoter, particularly a proximal promoter) or enhancer or silencer sequence.

[0052] In one aspect, the present invention provides an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein and a guide RNA that targets a DNA molecule encoding a gene product in a cell, whereby the guide RNA targets the DNA molecule encoding the gene product and the Cas protein cleaves the DNA molecule encoding the gene product, thereby altering expression of the gene product; the Cas protein and guide RNA do not occur together in nature. The present invention encompasses a guide RNA comprising a guide sequence fused to a tracr sequence. In one embodiment of the present invention, the Cas protein is a type II CRISPR-Cas protein, and in a preferred embodiment, the Cas protein is a Cas9 protein. The present invention further encompasses a Cas9 protein that has been codon-optimized for expression in eukaryotic cells. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the present invention, expression of a gene product is reduced.

[0053] In another aspect, the present invention provides an engineered, non-naturally occurring vector system comprising one or more vectors, the vectors comprising a first regulatory element operably linked to a CRISPR-Cas system guide RNA that targets a DNA molecule encoding a gene product, and a second regulatory element operably linked to a Cas protein. Components (a) and (b) can be located on the same or different vectors of the system. The guide RNA targets the DNA molecule encoding the gene product in a cell, and the Cas protein cleaves the DNA molecule encoding the gene product, thereby altering expression of the gene product; the Cas protein and guide RNA do not occur together in nature. The present invention encompasses guide RNAs comprising a guide sequence fused to a tracr sequence. In one embodiment of the present invention, the Cas protein is a type II CRISPR-Cas protein, and in a preferred embodiment, the Cas protein is a Cas9 protein. The present invention further encompasses Cas proteins that are codon-optimized for expression in eukaryotic cells. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the present invention, expression of a gene product is reduced.

[0054] In one aspect, the present invention provides a vector system, comprising one or more vectors.In some embodiments, the system comprises: (a) a first regulatory element, which is 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 (when expressed, the guide sequence directs the sequence-specific binding of CRISPR complex to target sequence in eukaryotic cells, and the CRISPR complex comprises (1) the guide sequence hybridized to target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence); and (b) a second regulatory element, which is operably linked to the enzyme coding sequence encoding the CRISPR enzyme, which comprises a nuclear localization sequence; component (a) and (b) are 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, and each of the two or more guide sequences, when expressed, directs the sequence-specific binding of CRISPR complexes to different target sequences in eukaryotic cells.In some embodiments, the system comprises a third regulatory element, for example, a tracr sequence under the control of a polymerase III promoter.In some embodiments, the tracr sequence shows at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.Determining optimal alignment is within the skill of those skilled in the art.For example, there are publicly available 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 being bound by theory, although nuclear localization sequences are not required for CRISPR complex activity in eukaryotes, it is believed that including such sequences improves the activity of the system, particularly for 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 Streptococcus pneumoniae (S. pneumoniae), Streptococcus pyogenes (S. pyogenes), or S. thermophilus Cas9, and may include mutant Cas9s 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 eukaryotic cells. In some embodiments, the CRISPR enzyme directs one or two strand cleavage at the location of the target sequence. 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, or 25 nucleotides, or 10-30, or 15-25, or 15-20 nucleotides in length. Generally, and throughout this specification, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules that contain one or more free ends but no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and various other 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, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which viral-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses).Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Some vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Common expression vectors useful in recombinant DNA technology are often in the form of plasmids.

[0055] A recombinant expression vector can contain a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, meaning that the recombinant expression vector can be selected based on the host cell to be used for expression and contains one or more regulatory elements operably linked to the nucleic acid sequence to be expressed. In the context of a recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is linked to a regulatory element 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).

[0056] The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, e.g., 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 cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired target tissue, e.g., muscle, nerve cells, bone, skin, blood, specific organs (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). The regulatory elements can also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue- or cell type-specific. In some embodiments, the vector comprises one or more PolIII promoters (e.g., 1, 2, 3, 4, 5, or more PolI promoters), one or more PolII promoters (e.g., 1, 2, 3, 4, 5, or more PolII promoters), one or more PolI promoters (e.g., 1, 2, 3, 4, 5, or more PolI promoters), or a combination thereof. Examples of PolIII 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 β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. The term "regulatory element" also encompasses enhancer elements, such as the WPRE; the CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). Those skilled in the art will recognize that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired expression level, and the like. The vector can be introduced into a host cell to thereby produce proteins, including transcripts, fusion proteins, or peptides, encoded by the nucleic acids described herein (e.g., clustered regularly interspaced short repeats (CRISPR) transcripts, proteins, enzymes, mutants thereof, fusion proteins thereof, etc.).

[0057] Advantageous vectors include lentiviruses and adeno-associated viruses, and such vector types can also be selected for targeting specific cell types.

[0058] In one aspect, the present 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 (the guide sequence, when expressed, directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, the CRISPR complex comprising a CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence and (2) the tracr mate sequence hybridized to the tracr sequence); and / or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme comprising a nuclear localization sequence. In some embodiments, the host cell comprises components (a) and (b). In some embodiments, components (a), (b), or (a) and (b) are stably integrated into the genome of the host eukaryotic cell. In some embodiments, component (a) further comprises a 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, each of which, when expressed, directs sequence-specific binding of the CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the eukaryotic host cell further comprises a third regulatory element operably linked to the tracr sequence, such as a polymerase III promoter. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells. 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, or 25 nucleotides, or 10-30, or 15-25, or 15-20 nucleotides in length. In one 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 another aspect, the invention provides a eukaryotic organism, preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments. In some embodiments of these aspects, the organism may be an animal, e.g., a mammal. The organism may also be an arthropod, e.g., an insect. The organism may also be a plant. Furthermore, the organism may be a fungus.

[0059] In one aspect, the present 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 use of 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 (the guide sequence, when expressed, directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, the CRISPR complex comprising (1) a guide sequence hybridized to the target sequence, and (2) a CRISPR enzyme complexed with the tracr mate sequence hybridized to the tracr sequence); and / or (b) a second regulatory element operably linked to an enzyme coding sequence encoding the CRISPR enzyme comprising a nuclear localization sequence. In some embodiments, the kit comprises components (a) and (b) on the same or different vectors of the system. In some embodiments, component (a) further comprises a 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, each of which, when expressed, directs sequence-specific binding of the CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system further comprises a third regulatory element operably linked to the tracr sequence, such as a polymerase III promoter. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% 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 strong enough to drive the accumulation of a detectable amount of the CRISPR enzyme 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 mutant Cas9s from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the CRISPR enzyme directs one or two strand cleavage 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, or 25 nucleotides, or 10-30, 15-25, or 15-20 nucleotides in length.

[0060] In one aspect, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises binding a CRISPR complex to a target polynucleotide to cause cleavage of the 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 the target polynucleotide, the guide sequence in turn being bound to a tracr mate sequence hybridized to a tracr sequence. In some embodiments, the cleavage comprises cleaving one or both strands at the location of the target sequence by the CRISPR enzyme. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides in the target polynucleotide. In some embodiments, the 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 the 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. In some embodiments, the vectors are delivered into the eukaryotic cell in a subject. In some embodiments, the modification is performed in the eukaryotic cell in cell culture. In some embodiments, the method further comprises isolating the eukaryotic cell from a subject before the modification. In some embodiments, the method further comprises returning the eukaryotic cell and / or cells derived therefrom to the subject.

[0061] In one aspect, the present invention provides a method for modifying the expression of polynucleotide in eukaryotic cells.In some embodiments, the method comprises: allowing CRISPR complex to bind to polynucleotide, whereby said binding causes the expression of said polynucleotide to increase or decrease; said CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence in said polynucleotide, and said guide sequence is then linked to a tracr mate sequence that hybridizes to a tracr sequence.In some embodiments, the method further comprises delivering one or more vectors into said eukaryotic cell, and said one or more vectors drive the expression of one or more of CRISPR enzyme, the guide sequence that is linked to a tracr mate sequence, and the tracr sequence.

[0062] In one aspect, the present invention provides a method for generating a model eukaryotic cell containing a mutated disease gene. In some embodiments, the disease gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method includes (a) introducing one or more vectors into a eukaryotic cell, where 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) binding a CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide within the disease gene, where the CRISPR complex comprises (1) a guide sequence hybridized to a target sequence within the target polynucleotide, and (2) a CRISPR enzyme complexed with a tracr mate sequence hybridized to a tracr sequence, thereby generating a model eukaryotic cell containing a mutated disease gene. In some embodiments, the cleavage includes cleavage of one or both strands at the location of the target sequence by the CRISPR enzyme. In some embodiments, the cleavage results in decreased transcription of the target gene. In some embodiments, the method further comprises repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides in the target polynucleotide. In some embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.

[0063] In one aspect, the present invention provides a method for developing a bioactive agent that modulates a cell signaling event associated with a disease gene. In some embodiments, the disease gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method includes: (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 indicates a reduction or increase in a cell signaling event associated with the mutation in the disease gene, thereby developing the bioactive agent that modulates the cell signaling event associated with the disease gene.

[0064] In one aspect, the present invention provides a recombinant polynucleotide comprising a guide sequence upstream of a tracr mate sequence, wherein the guide sequence, when expressed, directs the sequence-specific binding of a CRISPR complex to the corresponding target sequence present in eukaryotic cells.In some embodiments, the target sequence is a viral sequence present in eukaryotic cells.In some embodiments, the target sequence is a proto-oncogene or an oncogene.

[0065] In one aspect, the present invention provides a method for selecting one or more cells by introducing one or more mutations into a gene in one or more cells, the method comprising: introducing one or more vectors into the cells (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 an editing template; the editing template comprises one or more mutations that prevent CRISPR enzyme cleavage); allowing the editing template to undergo homologous recombination with a target polynucleotide in the cell to be selected; and allowing a CRISPR complex to bind to the target polynucleotide, resulting in cleavage of the target polynucleotide within the gene (the CRISPR complex comprises (1) a guide sequence hybridized to a target sequence within the target polynucleotide, and (2) a tracr mate sequence hybridized to the tracr sequence, wherein binding of the CRISPR complex to the target polynucleotide induces cell death), thereby enabling the selection of one or more cells into which one or more mutations have been introduced. In a preferred embodiment, the CRISPR enzyme is Cas9. In another preferred embodiment of the present invention, the cell to be selected may be a eukaryotic cell. Aspects of the present invention allow for the selection of defined cells without requiring a selectable marker or a two-step process that may involve a counterselection system.

[0066] In another aspect, the invention encompasses a CRISPR-Cas9 (S. pyogenes) system having an X-ray diffraction pattern corresponding to or obtained from a crystal having a structure defined by the coordinates of any or all of the above and / or the crystal structure table in Example 8 (CRISPR-cas9 crystal structure) or as further described in Example 12.

[0067] In a further aspect, the invention comprises a computer-aided method for identifying or designing potential compounds that fit within or bind to a CRISPR-Cas9 system or functional portion thereof, or vice versa (a computer-aided method for identifying or designing a potential CRISPR-Cas9 system or functional portion thereof that binds to a desired compound) or a computer-aided method for identifying or designing a potential CRISPR-Cas9 system (e.g., with respect to predicted areas of the CRISPR-Cas9 system that can be manipulated, e.g., based on crystal structure data, or based on data of Cas9 orthologues, or with respect to where functional groups, e.g., activators or repressors, can be attached to the CRISPR-Cas9 system, or with respect to Cas9 truncation, or with respect to designing a nickase), said method comprising: using a computer system, e.g., a programmed computer including a processor, a data storage system, input devices, and output devices; (a) inputting into the programmed computer via the input device, for example, data including three-dimensional coordinates of a subset of atoms from or related to a CRISPR-Cas9 crystal structure in a CRISPR-Cas9 system binding domain or, alternatively or additionally, in a domain that varies based on variance between Cas9 orthologs, or related to Cas9, or related to a nickase, or related to a functional group, optionally together with structural information from a CRISPR-Cas9 system complex, thereby generating a dataset; (b) using the processor, comparing the dataset to a computer database of structures stored in the computer data storage system, e.g., structures of compounds that bind or putatively bind to, or are desired to bind to, a CRISPR-Cas9 system, or for Cas9 orthologs (e.g., as Cas9 or for domains or regions that vary between Cas9 orthologs), or for CRISPR-Cas9 crystal structures, or for nickases, or for functional groups; (c) using computational methods to select from said database a structure, e.g., a CRISPR-Cas9 structure that can bind to a desired structure, a desired structure that can bind to a CRISPR-Cas9 structure, e.g., a portion of a CRISPR-Cas9 system that can be engineered based on data from other portions of the CRISPR-Cas9 crystal structure and / or from a Cas9 ortholog, a truncated Cas9, a novel nickase or a particular functional group, or a functional group or functional group-CRISPR-Cas9 system; (d) using a computational method to construct a model of the selection structure; and (e) outputting the selection structure to the output device; and optionally synthesizing one or more of the selected structures; and further optionally, testing said synthetic selection structure as or in a CRISPR-Cas9 system; Alternatively, the method may include providing coordinates of at least two atoms of a CRISPR-Cas9 crystal structure, e.g., coordinates of at least two atoms in a crystal structure table herein of a CRISPR-Cas9 crystal structure, or coordinates of at least a subdomain of a CRISPR-Cas9 crystal structure ("selected coordinates"); e.g., providing candidate structures comprising a binding molecule of a CRISPR-Cas9 system or a binding molecule of a portion thereof, which can be engineered based on data from other portions of the CRISPR-Cas9 crystal structure and / or from structures of Cas9 orthologs or functional groups; and selecting the candidate structures. and obtaining as its output product data including a CRISPR-Cas9 structure that fits the coordinates and thereby can bind to a desired structure, a desired structure that can bind to a CRISPR-Cas9 structure, a portion of a CRISPR-Cas9 system that can be engineered, a truncated Cas9, a novel nickase, or a particular functional group, or a location for attaching a functional group or functional group-CRISPR-Cas9 system; and optionally synthesizing a compound from said product data, and further optionally testing said synthesized compound as or in a CRISPR-Cas9 system.

[0068] Testing can include analyzing the CRISPR-Cas9 system obtained from the synthetic selection construct, for example, for binding or performance of a desired function.

[0069] The output of the above method can include data transmission, e.g., transmission of information via telecommunications, telephone, videoconferencing, mass communication, e.g., a presentation, e.g., a computer presentation (e.g., POWERPOINT), the Internet, email, written communication, e.g., a computer program (e.g., WORD) document, etc. Accordingly, the present invention also encompasses computer-readable media containing atomic coordinate data from the crystal structure tables and / or figures herein, which data define the three-dimensional structure of CRISPR-Cas9 or at least one subdomain thereof, or structure factor data for CRISPR-Cas9, which structure factor data can be derived from the atomic coordinate data from the crystal structure tables and / or figures herein. The computer-readable media can also contain any of the data of the above method. The invention further encompasses methods, computer systems, and methods for generating or implementing rational designs as described above, containing either atomic coordinate data according to the crystal structure tables and / or figures herein, which data define the three-dimensional structure of CRISPR-Cas9, or at least one subdomain thereof, or structure factor data for CRISPR-Cas9, which structure factor data is derivable from the atomic coordinate data in the crystal structure tables and / or figures herein. The invention further encompasses methods of performing operations, including providing to a user a computer system or medium, or the three-dimensional structure of CRISPR-Cas9 or at least one subdomain thereof, or structure factor data for CRISPR-Cas9, which structure is set forth in the crystal structure tables and / or figures herein, and which structure factor data is derivable from the atomic coordinate data in the crystal structure tables and / or figures herein, or the computer medium herein, or data transmission herein.

[0070] A "binding site" or "active site" comprises or essentially consists of a site in the binding cavity or region (e.g., an atom, functional group or multiple such atoms and / or groups of an amino acid residue) that can bind to a compound involved in binding, e.g., a nucleic acid molecule.

[0071] "Fitting" means determining, by automatic or semi-automatic means, the interactions between one or more atoms of a candidate molecule and at least one atom of a structure of the invention, and calculating the degree to which such interactions are stable. Interactions include attractive and repulsive forces arising from charge, steric considerations, etc. Various methods of computer-based fitting are further described.

[0072] "Root mean square (or rms) deviation" means the square root of the arithmetic mean of the squares of the deviations from the mean.

[0073] "Computer system" refers to the hardware, software, and data storage means used to analyze atomic coordinate data. The minimum hardware means of the computer-based systems of the present invention typically includes a central processing unit (CPU), input means, output means, and data storage means. Desirably, a display or monitor is provided for visualizing the structural data. The data storage means can be RAM or a means for accessing the computer-readable medium of the present invention. Examples of such systems are computers and tablet devices running Unix, Windows, or Apple operating systems.

[0074] "Computer-readable medium" refers to any one or more media that can be read and accessed directly or indirectly by a computer, such that the media is suitable for use in a computer system. Such media include, but are not limited to, magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tape; optical storage media, such as optical disks or CD-ROMs; electrical storage media, such as RAM and ROM; thumb drive devices; cloud storage devices, and hybrids of these categories, such as magnetic / optical storage media.

[0075] In certain embodiments of the present invention, conformational variations in the crystal structure of a CRISPR-Cas9 system, or of components of a CRISPR-Cas9 system, provide important and critical information about the flexibility or mobility of protein structural regions relative to nucleotide (RNA or DNA) structural regions that may be important for CRISPR-Cas system function. The structural information provided in this application for Cas9 as a CRISPR enzyme (e.g., S. pyogenes Cas9) can be used to further engineer and optimize CRISPR-Cas systems, and can be extrapolated to explore structure-function relationships in other CRISPR enzyme systems, such as other type II CRISPR enzyme systems.

[0076] The present invention particularly encompasses the CRISPR-Cas enzyme system of the present invention and the modification guide of the present invention, and the CRISPR enzyme is also associated with a functional domain.In particular, the CRISPR enzyme comprises one or more mutations that convert it into a DNA-binding protein that can recruit, attach, insert, or attach functional domains that exhibit desired functions.In some embodiments, the CRISPR enzyme comprises one or more mutations, including, but not limited to, D10A, E762A, H840A, N854A, N863A, or D986A (based on the amino acid position numbering of Streptococcus pyogenes (S. pyogenes) Cas9), and / or one or more mutations are present in the RuvC1 or HNH domain of the CRISPR enzyme, or other mutations described herein. In some embodiments, the CRISPR enzyme has one or more mutations in the catalytic domain, the tracr mate sequence hybridizes to the tracr sequence when transcribed, the guide sequence directs the sequence-specific binding of the CRISPR complex to the target sequence, and the enzyme further comprises a functional domain. In some embodiments, the mutation at N580 in the SaCas9 protein is preferred. In particular, instead of the mutation in SaCas9, the mutation corresponding to H840 in SpCas9 is preferred. In some embodiments, the mutations at D10 and N580 in SaCas9 are preferred. In some embodiments, the N580 mutation can be N580A in the SaCas9 protein. Without being bound by theory, this is thought to be a more predictable mutation for protein function than the equivalent H840A, which can change binding behavior.

[0077] The structural information provided herein allows for investigation of sgRNA (or chimeric RNA) interactions with target DNA and CRISPR enzymes (e.g., Cas9), allowing for engineering or modification of sgRNA structure to optimize the functionality of the entire CRISPR-Cas system. For example, insertion of an adaptor protein capable of binding to RNA can extend the loop of the sgRNA without colliding with the Cas9 protein. These adaptor proteins can further recruit effector proteins or fusions containing one or more functional domains.

[0078] In some preferred embodiments, the functional domain is a transcription activation domain, preferably VP64. In some embodiments, the functional domain is a transcription repression domain, preferably KRAB. In some embodiments, the transcription repression domain is SID or a concatemer of SIDs (e.g., SID4X). In some embodiments, the functional domain is an epigenetic modification domain, thereby providing an epigenetic modification enzyme. In some embodiments, the functional domain is an activation domain, which may be a P65 activation domain.

[0079] Aspects of the present invention include non-naturally occurring or engineered compositions that include a guide RNA (sgRNA) that includes a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, and a CRISPR enzyme that may include at least one or more nuclear localization sequences, where the CRISPR enzyme includes two or more mutations such that the enzyme has altered or reduced nuclease activity compared to the wild-type enzyme, and at least one loop of the sgRNA is modified with the insertion of a distinct RNA molecule that binds to one or more adaptor proteins, where the adaptor proteins further recruit one or more heterologous functional domains. In one embodiment of the present invention, the CRISPR enzyme includes two or more mutations in residues selected from the group consisting of D10, E762, H840, N854, N863, or D986. In a further embodiment, the CRISPR enzyme includes two or more mutations selected from the group including D10A, E762A, H840A, N854A, N863A, or D986A. As described above, N580, particularly N580A, of the SaCas9 protein is particularly useful in SaCas9. In another embodiment, the functional domain is a transcriptional activation domain, e.g., VP64. In another embodiment, the functional domain is a transcriptional repressor domain, e.g., a KRAB domain, a SID domain, or a SID4X domain. In an embodiment of the present invention, the one or more heterologous functional domains have one or more activities selected from the group consisting of methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. In a further embodiment of the present invention, the cell is a eukaryotic cell, a mammalian cell, or a human cell. In further embodiments, the adaptor protein is selected from the group consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1.In another embodiment, at least one loop of the sgRNA is a tetraloop and / or loop 2. One aspect of the invention includes a method of modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell any of the compositions described herein.

[0080] One aspect of the present invention is that the elements are contained in a single composition or in separate compositions that can be advantageously applied to a host to induce a functional effect on the genome level.

[0081] Generally, the sgRNA is modified to provide a specific binding site (e.g., an aptamer) for an adaptor protein containing one or more functional domains to be bound (e.g., via a fusion protein). The modified sgRNA is modified so that, once the sgRNA forms a CRISPR complex (i.e., binding of the sgRNA and CRISPR enzyme to the target), the adaptor protein binds and the functional domain on the adaptor protein is positioned in a spatial orientation favorable for the assigned function to be effective. For example, if the functional domain is a transcriptional activator (e.g., VP64 or p65), the transcriptional activator is positioned in a spatial orientation that allows it to affect transcription of the target. Similarly, a transcriptional repressor is advantageously positioned to affect transcription of the target, and a nuclease (e.g., Fok1) is advantageously positioned to cleave or partially cleave the target.

[0082] Those skilled in the art will understand that modifications of the sgRNA that allow for binding of the adaptor+functional domain but do not allow for proper positioning of the adaptor+functional domain (e.g., due to steric hindrance within the three-dimensional structure of the CRISPR complex) are unintended modifications. One or more modified sgRNAs can be modified in the tetraloop, stem-loop 1, stem-loop 2, or stem-loop 3 as described herein, preferably in either the tetraloop or stem-loop 2, and most preferably in both the tetraloop and stem-loop 2.

[0083] As described herein, the functional domain can be, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switch (e.g., light-inducible).In some cases, it is advantageous to further provide at least one NLS.In some cases, it is advantageous to position the NLS at the N-terminus.When two or more functional domains are included, the functional domains can be the same or different.

[0084] The sgRNA can be designed to contain multiple binding recognition sites (e.g., aptamers) specific for the same or different adaptor proteins. The sgRNA can be designed to bind to the promoter region between -1000 and +1 nucleic acids upstream, preferably -200 nucleic acids upstream, of the transcription start site (i.e., TSS). This positioning improves functional domains that affect gene activation (e.g., transcriptional activators) or gene inhibition (e.g., transcriptional repressors). The modified sgRNAs can be one or more (e.g., at least one sgRNA, at least two sgRNAs, at least five sgRNAs, at least 10 sgRNAs, at least 20 sgRNAs, at least 30 sgRNAs, or at least 50 sgRNAs) targeted to one or more target loci included in the composition.

[0085] Furthermore, CRISPR enzymes with reduced nuclease activity are most effective when nuclease activity is inactivated (e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% nuclease inactivation compared to the wild-type enzyme; or in other words, advantageously, a Cas9 or CRISPR enzyme with about 0% of the nuclease activity of a non-mutated or wild-type Cas9 or CRISPR enzyme, or about 3%, about 5%, or about 10% or less of the nuclease activity of a non-mutated or wild-type Cas9 or CRISPR enzyme). This is possible by introducing mutations into the RuvC and HNH nuclease domains of SpCas9 and its orthologs. For example, mutations in residues selected from the group consisting of D10, E762, H840, N854, N863, or D986 are utilized, more preferably one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A, or D986A. A preferred pair of mutations is D10A and H840A, more preferably D10A and N863A, in SpCas9 and its orthologs. In some embodiments, N580A in the SaCas9 protein can be used as described herein.

[0086] The inactivated CRISPR enzyme may be associated (e.g., via a fusion protein) with one or more functional domains, such as those described herein for modified sgRNA adapter proteins, including, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switch (e.g., light-inducible). Preferred domains are Fok1, VP64, P65, HSF1, and MyoD1. In the event that Fok1 is provided, it is advantageous to design the sgRNA to provide multiple Fok1 functional domains to enable functional dimerization and provide appropriate spacing for functional use (Fok1), as specifically described in Tsai et al. Nature Biotechnology, Vol. 32, Number 6, June 2014). Adapter proteins may utilize known linkers to attach such functional domains. In some cases, it is advantageous to further provide at least one NLS. In some cases, it is advantageous to position the NLS at the N-terminus. When two or more functional domains are included, the functional domains may be the same or different.

[0087] Generally, the positioning of one or more functional domains on inactivated CRISPR enzyme allows the functional domain to be precisely positioned in space to affect the target by the attributed functional effect.For example, when the functional domain is a transcription activator (for example, VP64 or p65), the transcription activator is arranged in a space orientation that allows it to affect the transcription of the target.Similarly, the transcription repressor is advantageously positioned to affect the transcription of the target, and the nuclease (for example, Fok1) is advantageously positioned to cleave or partially cleave the target.This can include positions other than the N / C terminus of CRISPR enzyme.

[0088] Based on crystal structure experiments, the present applicants have identified that it is advantageous to position a functional domain in the Rec1 domain, Rec2 domain, HNH domain, or PI domain of the SpCas9 protein, or any orthologs corresponding to these domains. Positioning a functional domain in the Rec1 domain or Rec2 domain of the SpCas9 protein, or any orthologs corresponding to these domains, may be preferred in some cases. Positioning a functional domain in the Rec1 domain at position 553, the Rec1 domain at position 575, the Rec2 domain or a substitution thereof at any position between 175 and 306, the HNH domain or a substitution thereof at any position between 715 and 901, or the PI domain at position 1153, or any orthologs corresponding to these domains, may be preferred in some cases. The Fok1 functional domain can be attached at the N-terminus. When two or more functional domains are included, the functional domains may be the same or different.

[0089] The adaptor protein can be any number of proteins that bind to the aptamer or recognition site introduced into the modified sgRNA and allow for the appropriate positioning of one or more functional domains that affect the target function once the sgRNA is incorporated into the CRISPR complex. As described in detail in this application, such a protein can be a coat protein, preferably a bacteriophage coat protein. The functional domain associated with such an adaptor protein (e.g., in the form of a fusion protein) can include, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switch (e.g., light-inducible). Preferred domains are Fok1, VP64, P65, HSF1, and MyoD1. In the event that the functional domain is a transcription activator or transcription repressor, it is advantageous to further provide at least an NLS, preferably at the N-terminus. When two or more functional domains are included, the functional domains can be the same or different. Adaptor proteins may utilize known linkers to attach such functional domains.

[0090] Therefore, the modified sgRNA, the inactivated CRISPR enzyme (with or without functional domains), and the binding protein having one or more functional domains can each be individually included in a composition and administered to the host individually or collectively. Alternatively, these components can be provided in a single composition for administration to the host. Administration to the host can be carried out via a viral vector (e.g., a lentiviral vector, an adenoviral vector, an AAV vector) known to those skilled in the art for delivery to the host or described herein. As described herein, the use of different selection markers (e.g., for lentiviral sgRNA selection) and the concentration of the sgRNA (e.g., depending on whether multiple sgRNAs are used) can be advantageous in eliciting improved effects.

[0091] Based on this concept, some variations are suitable for inducing genomic locus events, such as DNA cleavage, gene activation, or gene inactivation.Using the provided compositions, those skilled in the art can advantageously and specifically target single or multiple loci with the same or different functional domains to induce one or more genomic locus events.The compositions can be applied in a wide range of methods for screening in cellular libraries and in vivo function modeling (e.g., identifying gene activation and function of lincRNA; gain-of-function modeling; loss-of-function modeling; using the compositions of the present invention to establish cell lines and transgenic animals for optimization and screening purposes).

[0092] The present invention encompasses the use of the compositions of the present invention to establish and utilize conditional or inducible CRISPR transgenic cells / animals (see, e.g., Platt et al., Cell (2014), http: / / dx.doi.org / 10.1016 / j.cell.2014.09.014, or PCT patent application publications cited herein, e.g., International Publication No. WO 2014 / 093622 (PCT / US2013 / 074667), which are not believed to precede this invention or this application). For example, the target cell conditionally or inducibly contains a CRISPR enzyme (e.g., Cas9) and / or a conditionally or inducibly contained adapter protein (e.g., in the form of a Cre-dependent construct), and upon expression of the vector introduced into the target cell, the vector expresses an inducer or condition for CRISPR enzyme (e.g., Cas9) expression and / or adapter expression in the target cell. By applying the teachings and compositions of the present invention to known methods for generating CRISPR complexes, inducible genomic events influenced by functional domains are also an aspect of the present invention. Another example of this is the generation of CRISPR knock-in / conditional transgenic animals (e.g., mice containing a Lox-Stop-polyA-Lox (LSL) cassette) and the subsequent delivery of one or more compositions providing one or more modified sgRNAs (e.g., -200 nucleotides relative to the TSS of a target gene of interest for gene activation purposes) (e.g., sgRNAs modified with one or more aptamers recognized by coat proteins, e.g., MS2), one or more adaptor proteins (e.g., MS2 binding proteins bound to one or more VP64s) described herein, and a means of inducing the conditional animal (e.g., Cre recombinase for inducible Cas9 expression). Alternatively, the adaptor protein can be provided as a conditional or inducible element by a conditional or inducible CRISPR enzyme, advantageously providing a useful model for screening purposes, requiring only minimal design and administration of specific sgRNAs for multiple applications.

[0093] In some embodiments, the CRISPR enzyme is selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphingobacterium, Sphingobacterium sp., Sphingobacterium spp ... Cas9 orthologs from genera belonging to the group consisting of Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, and Campylobacter, where Cas contains a helical domain 2 truncation.

[0094] In one aspect, the invention provides a composition, method, or use as described herein, wherein the helical domain 2 truncation is replaced by one or more sets of flexible glycine-serine (GlyGlyGlySer) or rigid alpha-helical linkers (Ala(GluAlaAlaAlaLys)Ala) in groups of 3, 6, 9, or 12 repeats to replace helical domain 2, potentially for structural stabilization and / or to assist in retaining Cas9:sgRNA specificity.

[0095] In one aspect, the invention provides a composition, method, or use as described herein, wherein the CRISPR enzyme is a Cas, e.g., SpCas9 or SaCas9.

[0096] In one embodiment, the present invention provides a non-naturally occurring CRISPR enzyme, wherein the HD2 domain is truncated. The CRISPR enzyme may be Cas9, such as SpCas9 or SaCas9.

[0097] In one embodiment, the invention provides a CRISPR enzyme, wherein the truncation is a replacement of the HD2 domain, e.g., the truncation is a replacement of the HD2 domain with a linker, e.g., a flexible linker; e.g., a GlySer linker.

[0098] In one embodiment, the present invention provides a chimeric three-component CRISPR enzyme, comprising N'- and C'-terminal components from a first CRISPR enzyme and an internal component from a second CRISPR enzyme, wherein the second CRISPR enzyme is an ortholog of the first CRISPR enzyme; for example, the first and second CRISPR enzymes each comprise Cas9, such as SpCas9 or SaCas9 or StCas9, for example, St3Cas9. In some embodiments, the CRISPR enzyme described herein may comprise an internal component from the first CRISPR enzyme replaced by an internal component from the second CRISPR enzyme, wherein the internal components are the same or different. In one embodiment of the present invention, the second CRISPR enzyme is a SaCRISPR enzyme, a StCRISPR enzyme, or a St3CRISPR enzyme. The present invention contemplates the compositions, methods, systems, or uses described herein, wherein the CRISPR enzyme is chimeric or truncated as described herein.

[0099] Accordingly, it is not the object of the present invention to encompass any previously known products, methods of making the products, or methods of using the products, and therefore, applicants reserve the right to, and hereby disclose, a disclaimer of any previously known products, methods of making the products, or methods of using the products. It is further noted that the present invention does not encompass within its scope any products, methods, or methods of making or using the products that do not meet the description and enablement requirements of the USPTO (35 U.S.C. Section 112, first paragraph) or the EPO (European Patent Convention, Article 83), and therefore, applicants reserve the right to, and hereby disclose, a disclaimer of any previously described products, methods of making the products, or methods of using the products.

[0100] It is noted that in this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like, may have the meaning ascribed to them in U.S. Patent Law; for example, they may mean "includes," "included," "including," and the like; and terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent Law, for example, they allow for elements not expressly recited, but exclude elements found in the prior art or that affect a basic or novel characteristic of the invention. Nothing herein should be construed as a commitment.

[0101] These and other embodiments are disclosed or are obvious from and encompassed by the following detailed description.

[0102] 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. [Brief explanation of the drawings]

[0103] [Figure 1] A schematic model of the CRISPR system is shown. 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) immediately upstream of a required 5'-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-strand break (DSB) (red triangle) approximately 3 bp upstream of the PAM. [Figure 2A-F] Exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing nuclear localization and CRISPR activity are presented. [Figure 3A-D] 1 shows the results of an assessment of SpCas9 specificity for exemplary targets. [Figure 4A-G] An exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells are presented. [Figure 5] A table of protospacer sequences is provided, and modification efficiency results for protospacer targets and corresponding PAMs designed based on exemplary S. pyogenes and S. thermophilus CRISPR systems for loci in the human and mouse genomes are summarized. Cells were transfected with Cas9 and either pre-crRNA / tracrRNA or chimeric RNA and analyzed 72 hours post-transfection. Percent indels were calculated based on Surveyor assay results from the indicated cell lines (N=3 for all protospacer targets; errors are standard error of the mean (SEM) where ND indicates not detectable using the Surveyor assay and NT indicates not tested in this study). [Figure 6A-C] 1 shows a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting. [Figure 7]Figure 1 shows a schematic diagram of the surveyor nuclease assay for the detection of double-strand break-induced microinsertions and deletions. [Figure 8A-B] 1 shows an exemplary bicistronic expression vector for expression of CRISPR system elements in eukaryotic cells. [Figure 9A-C] Histogram of the distance between the adjacent S. pyogenes SF370 locus 1 PAM (NGG) (Figure 9A) and S. thermophilus LMD9 locus 2 PAM (NNAGAAW) (Figure 9B) in the human genome; as well as the distance for each PAM in chromosomes (Chr) (Figure 9C). [Figure 10A-C] 1 shows an exemplary CRISPR system, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing CRISPR activity. [Figure 11A-C] 1 shows an exemplary operation of the CRISPR system for targeting genomic loci in mammalian cells. [Figure 12A-B] 1 shows the results of Northern blot analysis of crRNA processing in mammalian cells. [Figure 13] Exemplary selection of protospacers in the human PVALB and mouse Th loci is shown. [Figure 14] 1 shows an exemplary protospacer and corresponding PAM sequence target of the S. thermophilus CRISPR system in the human EMX1 locus. [Figure 15] A table of sequences is provided for the primers and probes used in Surveyor, RFLP, genomic sequencing, and Northern blot assays. [Figures 16A-C] 1 shows an exemplary engineering of a CRISPR system with chimeric RNA and the results of a SURVEYOR assay for system activity in eukaryotic cells. [Figure 17A-B] 1 shows a graphical representation of the results of a SURVEYOR assay for CRISPR system activity in eukaryotic cells. [Figure 18]FIG. 1 shows an exemplary visualization of several Streptococcus pyogenes (S. pyogenes) Cas9 target sites in the human genome using the UCSC genome browser. [Figure 19A-D] A circular representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 20A-F] A linear representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 21A-D] Genome editing via homologous recombination. (a) Schematic of SpCas9 nickase with a D10A mutation in the RuvC I catalytic domain. (b) Schematic depicting homologous recombination (HR) at the human EMX1 locus using either a sense or antisense single-stranded oligonucleotide as the repair template. The upper red arrow indicates the sgRNA cleavage site; PCR primers for genotyping (Tables J and K) are shown as arrows in the right panel. (c) Sequence of the region modified by HR. d, SURVEYOR assay (n=3) for wild-type (wt) and nickase (D10A) SpCas9-mediated indels at the EMX1 target locus. Arrows indicate the positions of predicted fragment sizes. [Figure 22A-B] 1 shows a single vector design for SpCas9. [Figure 23A-M] A diagram showing the topology of the Cas9 protein is provided. Ribbon representations and various views of the CRISPR-cas complex crystal structure (A-I), chimeric RNA architecture from the crystal structure (J-K), a schematic diagram of interactions from the crystal structure (L), and a schematic diagram of topology from the crystal structure (M) are provided. [Figure 24A-C]Shown are the catalytic domain of SpCas9 from the crystal structure, a schematic diagram showing the sites of mutagenesis for new nickases (A), a schematic diagram showing the localization of sgRNA for testing double nicks (B), and Surveyor gel results showing one HNH mutant N854A that retains nickase activity, one HNH mutant (N863A) that exhibits nickase activity, and two RuvCIII mutants (H983A, D986A) that exhibit nickase activity (C). [Figure 25A-B] Shown are Surveyor gel results of SpCas9 truncation mutants from the crystal structure that retain cleavage activity (A) and a table showing amino acid truncations and flexible (GGGS) or rigid (A(EAAAK)) linker substitutions for the lanes of the gel in Figure 25A (B). [Figure 26A-B] Shown are SpCas9 sgRNAs from the crystal structure, including those mutated to investigate the contribution of defined bases or base groups to activity. [Figure 27A-C] Shown are truncations and creation of chimeric (S. pyogenes) Cas9s based on the crystal structure herein, including mutants for mapping essential functional domains (A), chimeras containing regions from S. thermophilus Cas9 (B), and designs for chemically induced dimerization of SpCas9 (C). [Figure 28] A photograph of a Cas9 crystal (0.2 mm) is shown. [Figure 29] A structural diagram showing the surface representation of Cas9 is shown; red, sgRNA; cyan, guide region of the sgRNA; gold, target DNA. [Figure 30A-D]The overall structure is shown. (A) Schematic of the domain organization of S. pyogenes Cas9 and the sgRNA:target DNA complex. (B) Ribbon representation of the Cas9-sgRNA-DNA complex. The irregular linker is shown as a dotted red line. (C) Surface representation of the Cas9-sgRNA-DNA complex. The active sites of the RuvC (D10A) and HNH (H840A) domains are indicated by dashed yellow circles. (D) Electrostatic surface potential of the Cas9-sgRNA-DNA complex. The HNH domain is omitted for clarity. Molecular graph images were generated using CueMol (see cuemol.org website). See also Figures 37 and 38. [Figure 31A-E] The REC lobe and PI domain are shown. (A) Structure of the REC lobe. The REC2 domain and bridge helix are colored dark gray and green, respectively. The REC1 domain is colored gray, and the repeat-interacting and anti-repeat-interacting regions are colored light blue and pink, respectively. The bound sgRNA:DNA is shown as a semi-transparent ribbon representation. (B) Schematic showing the location of SpCas9 truncations in the REC1 and REC2 domains. The bars on the right indicate indel mutations generated by the truncation mutants, as measured by the SURVEYOR assay (n=3, error bars indicate mean ± SEM, ND indicates not detectable). (C) Western blot showing the expression of truncation mutants in HEK293FT cells. (D) Structure of the PI domain. The bound sgRNA is shown as a semi-transparent ribbon representation. (E) Schematic showing wild-type SpCas9 and St3Cas9, chimeric Cas9, and SpCas9 PI domain truncation constructs. Cas9 assays for indel generation at target sites upstream of either NGG (left bar) or NGGNG (right bar) PAMs (n=3, error bars indicate mean ± SEM, ND indicates not detectable). See also Figures 39-41. [Figure 32A-F]The NUC lobe is shown. Helices are shown as cylinders, and beta sheets are shown as arrows. (A) Structure of the RuvC domain. The core structure of the RNase H fold core is highlighted in cyan. Active site residues are shown as stick models. (B) Structure of the T. thermophilus RuvC dimer in complex with a Holliday junction (PDB ID 4LD0). Two promoters are colored cyan and gray, respectively. (C) The sequence (top) illustrates Cas9 nicking targets on opposite strands of DNA. Targets 1 and 2 are separated by a distance of 4 bp between them. The heat map (bottom) shows the ability of each catalytic mutant to induce double-stranded (using either sgRNA1 or 2) or single-stranded cleavage (using both sgRNAs together only). Gray boxes: not assayed. (D) Indel formation by Cas9 nickase depends on the separation distance between the sgRNA pair (right panel). Separation distance is defined as the number of base pairs between the PAM-distal (5') ends of the guide sequences of a given sgRNA pair (n=3, error bars indicate mean ± SEM, ND indicates not detectable). (E) Structure of the HNH domain. The ββα-metallofold core structure is highlighted in magenta. Active site residues are shown as stick models. (F) Structure of the T4EndoVII dimer in complex with a Holliday junction (PDB ID 2QNC). The two promoters are colored pink and gray, respectively, and the ββα-metallofold core is highlighted in magenta. Bound Mg2+ ions are shown as orange spheres. [Figure 33A-D]The sgRNA and its target DNA are shown. (A) Schematic of the sgRNA:DNA complex. The guide and repeat regions of the crRNA sequence are colored light blue and blue, respectively. The tracrRNA sequence is colored red, and the linker region is colored cyan. The target DNA and tetraloop are colored yellow and black, respectively. The numbering of the 3' tail of the tracrRNA is shown against a red background. Watson-Crick and non-Watson-Crick base pairs are shown as black and gray lines, respectively. Irregular nucleotides are boxed with dotted lines. (B) Structure of the sgRNA:DNA complex. (C) Structure of the repeat:antirepeat duplex and three-way junction. Key interactions are shown as gray dotted lines. (D) Effect of sgRNA mutations on indel induction ability. The base change from the +83 sgRNA scaffold is indicated at each position, with the dotted line indicating the unchanged base (n=3, error bars indicate the mean ± SEM, p-values ​​are based on an unpaired Student's t-test, ND indicates not detectable). See also Figure 42. [Figure 34A-K] sgRNA:DNA recognition. (A) Schematic of sgRNA:DNA recognition by Cas9. Residues that interact with the sgRNA:DNA via the backbone are indicated in brackets. (B and C-K) Recognition of the guide (B), guide:DNA duplex (D), repeat (E), anti-repeat (F), three-way junction (G), stem-loop 1 (H), linker (I), stem-loop 2 (J), and stem-loop 3 (K). Hydrogen bonds and salt bridges are indicated as dotted lines. (C) Effect of Cas9 (top) and sgRNA (bottom) mutations on indel induction ability (n = 3, error bars indicate mean ± SEM, p-value is based on unpaired Student's t-test, ND indicates not detectable). [Fig. 35A-D]The structural flexibility of the complex is shown. (A) Structural comparison of Mol A and Mol B. In Mol A (left), the irregular linker between the RuvC and HNH domains is indicated by a dashed line. In Mol B (right), the irregular HNH domain is indicated by a dotted circle. The flexible connecting segments (α40 and α41) in the RuvC domain are highlighted in orange. (B) Superposition of Cas9 proteins in Mol A and Mol B. The two complexes are superimposed based on the core β-sheet of their RuvC domains. For clarity, the HNH domain and bound sgRNA:DNA are omitted. (C) Superposition of bound sgRNA:DNA in Mol A and Mol B. After superimposition of the two complexes in (B), the Cas9 protein is omitted to show the sgRNA:DNA. (D) Molecular surface of Cas9. For clarity, the HNH domain and bound sgRNA:DNA complex are omitted. Note that there are no direct contacts between the REC and NUC lobes, except for the interaction between the a2-α3 and β17-β18 loops. [Figure 36] A model of RNA-guided DNA cleavage by Cas9 is shown. [Figure 37] Electron density map: The 2mFO-DFC electron density map around the three-way junction is shown as a gray mesh (undulating at 2.5σ). [Figure 38A-C] We demonstrate that the di-cysteine ​​mutant (C80L / C574E) is functional in HEK293FT cells. (A) Schematic illustrating the location of the cysteine ​​mutations (C80L and C574E) in Cas9. (B) Sequence of the target site used to test the function of the C80L / C574E mutant of Cas9. (C) SURVEYOR nuclease assay (n=3) showing indels generated by either the wild type or the C80L / C574E mutant. [Figure 39] FIG. 1 shows a schematic illustrating the depiction of secondary structure elements of Cas9. [Figure 40A-B]Sequence alignment of Cas9 orthologs in families II-A and II-C is shown. Catalytic residues are indicated by red triangles. Critical arginine residues on the bridge helix are indicated by green triangles. The secondary structure of S. pyogenes Cas9 is shown above the sequence. This diagram was generated using TCoffee (Notredame et al., 2000) and ESPript (Gouet et al., 1999). Sp, S. pyogenes; Sm, Streptococcus mutans; St3, Streptococcus thermophilus CRISPR-3; St1, Streptococcus thermophilus CRISPR-1; Cj, Campylobacter jejuni; Mm, Neisseria meningiditis. [Figure 41] Sequence alignment of Cas9 orthologs in families II-A, II-B, and II-C is shown. Thirty-five Cas9 orthologs from families IIA, IIB, and IIC were aligned (BLOSUM62) and clustered (Jukes-Cantor model neighbor-joining method, using S. pyogenes Cas9 as an outgroup). The top bar indicates conservation by amino acid. In each line, black bars indicate residues with at least 75% consensus, and gray bars indicate non-conserved residues. [Figure 42] A comparison of the sgRNA:DNA heteroduplex with a reference A-form RNA duplex is shown. The sgRNA:DNA heteroduplex is superimposed on the A-form RNA duplex based on their phosphorus atoms. The A-form RNA duplex is colored dark gray. Nucleotides 51-97 of the sgRNA are omitted for clarity. [Figure 43] The DNA construct design of a previously studied dCas9 activator is shown. The activation domain is fused to the C-terminus of catalytically inactive dCas via a linker. An NLS is incorporated between Cas9 and VP64. [Figure 44]The illustration shows the insertion of the MS2 loop at the end of the tetraloop and loop 2 of the sgRNA. The MS2-VP64 fusion protein is recruited to these two loops. Together with dCas9, this results in the recruitment of the VP64 activation domain to the target DNA at the target locus. The inserted MS2 RNA stem loop is colored dark green. [Figure 45]

[0039] Figure 1 shows a graphical representation of the upregulation of Neurog2 expression in Neuro2A cells. Four different guide RNAs, with or without an Ms2 loop inserted into either the tetraloop or loop 2, or both, were tested in combination with dCas9 and MS2-vp64, dCas9-vp64 and MS2-vp64, or dCas9-vp64 alone. TL: MS2, MS2 loop insertion into sgRNA tetraloop; L2: MS2, MS2 loop insertion into loop 2 of the sgRNA. Colors indicate which protein-coding construct was co-transfected with the corresponding guide. [Figure 46] Figure 1 shows human ASCL1 upregulation by the Cas9-MS2 activator. [Figure 47] 1 shows human MYOD1 upregulation by the Cas9-MS2 activator. [Figure 48] 1 shows an illustration of orthogonal PP7 / MS2 gene targeting. In the schematic, sgRNAs targeting different gene loci are engineered with distinct RNA loops to recruit MS2-VP64 or PP7-SID4X, which activate and repress those loci, respectively. [Figure 49] The locations of transcription domain replacements and insertions in Cas9 are shown. The HNH domain is colored pink. The curved arrow indicates the movement of the HNH domain relative to the DNA (yellow) RNA (blue) duplex due to a conformational change. An A in a red circle indicates the first loop (AA G533) used for insertion of the transcription effector domain and its position relative to the target DNA. The third loop (K1153) for insertion of the transcription effector domain is indicated by a C in the red circle. [Figure 50]

[0033] Figure 1 shows the effect of sgRNA modifications on ASCL1 activation. The 3' MS2 and modified MS2 1.0 sgRNA architectures were tested for their ability to activate ASCL1. [Figure 51]

[0033] Figure 1 shows the effect of sgRNA-modified MYOD1 activation. The 3' MS2 and modified MS2 1.0 sgRNA architectures were tested for their ability to activate ASCL1. [Figure 52] 1 shows the effect of dCas9NLS and N863A modifications on ASCL1 activation. [Figure 53] 1 shows the effect of dCas9NLS and N863A modifications on MYOD1 activation. [Figure 54] Comparison of different NLS and point mutant dCas-VP64 architectures in combination with MS2 fused to individual or combined activation domains is shown. SV40: SV40NLS; a-imp: a-importin NLS; DV: dCas-SV40NLS-VP64; Mm: MS2-ASCL1TAD; MP: MS2-p65; MPH: MS2-p65-HSF1; MPm: MS2-p65-ASCL1TAD. All dCas9 proteins contain the D10A mutation and H840A (unless otherwise noted). [Figure 55] Comparison of different NLS and point mutant dCas-VP64 architectures in combination with MS2 fused to individual or combined activation domains is shown. SV40: SV40NLS; a-imp: a-importinNLS; DV: dCas-SV40NLS-VP64; Mm: MS2-MyodTAD; MP: MS2-p65; MPH: MS2-p65-HSF1; MPm: MS2-p65-MyodTAD. All dCas9 proteins contain the D10A mutation and H840A (unless otherwise noted). [Figure 56]Figure 1 shows the MS2 dual activator fusion protein for ASCL1 activation. Comparison of MS2-VP64 and MS2-p65 with and without an additional HSF1 activation domain fusion. The greatest relative improvement occurred with dCas9, which does not have its own activation domain. This improvement is particularly important for the future use of the system in multimodal transcriptional modulation, where transcriptional modulation occurs solely through the sgRNA and its aptamer protein, rather than through dCas9, allowing distinct guide sequences to target distinct functions. [Figure 57] 1 shows an MS2 dual activator fusion protein for MYOD1 activation. [Figure 58] Figure 1 shows the fold expression level activation by a single sgRNA guide sequence for 12 difficult-to-modulate genes. All activations demonstrated by the MS2-p65-HSF1 / SV40-dCas9-VP64 system. Guide localization is plotted against the TSS of each target. [Figure 59] 1 shows a plot of fold expression of the best guide sequence versus the delta Ct value from qPCR for that gene in control samples for the difficult targets listed above. [Figure 60] Multiplexed activation of 10 genes is shown. [Figure 61] 1 shows multiplexed activation of target genes. [Figure 62] Targeting of combinations of 2, 4, 6, 8, or 10 genes simultaneously using previously determined optimal single guides is shown. All experiments use NLS-dCAS(D10, H840A)-NLS-VP64 in combination with MS2-NLS-P65-HSF1. [Figure 63] Multiplexed activation panels of target genes are shown. [Figure 64A-F]Structure-guided design and optimization of the RNA-guided transcription activation complex is shown. a) The crystal structure of the Cas9-sgRNA-target DNA ternary complex (PDB ID: 4OO8) reveals occlusion of the N- and C-terminal fusion sites from the target DNA. The sgRNA tetraloop and stem-loop 2 largely do not contact Cas9 amino acid residues in this conformation and can be modified without altering the existing Cas9-sgRNA interaction. b) Diagram of the three-component transcription activation system (SAM): sgRNA2.0, the MS2-p65-HSF1 transcriptional transactivator, and the dCas9-VP64 fusion protein. The MS2 stem-loop addition to the sgRNA is highlighted in red. c) Design and optimization of the sgRNA scaffold for optimal recruitment of the MS2-VP64 transactivator. d) The MS2 stem-loop position within the sgRNA significantly influences transcription activation efficiency. e) Combinations of different activation domains act synergistically to further enhance the level of transcription activation. f, Addition of the HSF1 transactivation domain to MS2-p65 further increases the efficiency of transcriptional activation. All values ​​are mean ± SEM, n = 3. * indicates p < 0.05 based on Student's t-test. [Fig. 65A-D]Characterization of SAM-mediated gene activation and selection rules for efficient sgRNAs are shown. a) Fold activation of 12 different genes plotted against sgRNA localization. Distances are measured in bp relative to the TSS at +1. sgRNA1.0 with dCas9-VP64 (gray) and sgRNA2.0 with dCas9-VP64 and MS2-p65-HSF1 (blue). Arrows indicate sgRNA target sites with poor transcriptional activation. All values ​​are mean ± SEM, n = 3. b) Comparison of activation efficiencies achieved using dCas9-VP64 and a single sgRNA1.0 for the target gene; dCas9-VP64, a single sgRNA2.0 for the same target site as single sgRNA1.0, and MS2-p65-HSF1; and dCas9-VP64 and a mixture of eight sgRNAs targeting the same gene. c) Efficiency of target gene transcriptional activation as a function of its baseline expression level. Genes with higher basal levels of transcription show lower fold upregulation. For each target gene, baseline expression levels were measured using qPCR in GFP-transfected control cells and expressed as levels relative to GAPDH (lower fold expression compared to GAPDH on the x-axis). d, Correlation between gene activation efficiency and sgRNA targeting position in the proximal promoter region, expressed as distance to the TSS. Activation efficiency of each sgRNA for the same target gene is normalized to the maximally activating sgRNA. Proximity to the TSS positively correlates with target upregulation. The blue line indicates the median, and the boxes indicate the 25th and 75th percentiles. [Figure 66A-B] Figure 1 shows that SAM activates characterized and uncharacterized lincRNA transcripts. a) Fold activation of six lincRNAs plotted against sgRNA2.0 localization relative to the TSS. All values ​​are mean +-SEM, n=3. b) Correlation between sgRNA lincRNA activation efficiency and sgRNA target distance to the TSS. Activation efficiency of each sgRNA for the same target lincRNA is normalized to the maximally activating sgRNA. In contrast to coding genes, no significant correlation is observed. The blue line indicates the median, and the boxes indicate the 25th and 75th percentiles. [Figure 67A-E] Figure 1 shows simultaneous activation of endogenous genes using multiplexed sgRNA2.0 expression. a) Activation of individual genes with a single sgRNA2.0 carrying dCas9-VP64 and MS2-p65-HSF1. b) Simultaneous activation of 10 genes using a mixture of 10 sgRNA2.0s, each targeting a different gene. c) The relative efficiency of individual sgRNA2.0 activation varies depending on the target gene and the number of sgRNA2.0s targeting different genes. d) Effect of sgRNA dilution on gene activation efficiency. Results are plotted as the percentage of activation relative to the fold activation of a single undiluted sgRNA2.0 against the target gene. e) Correlation plot between the activation efficiency of a single 10-fold diluted sgRNA2.0 and the activation efficiency of the same sgRNA2.0 delivered within a mixture of sgRNA2.0s targeting 10 different genes. Performance across sgRNA dilutions significantly predicts performance in multiplexing, suggesting a guide-autonomous component to multiplexing behavior. All values ​​are mean +-SEM, n=3. [Figure 68A-E]Genome-scale lentiviral screen in mammalian cells using SAM is shown. a) Design of three lentiviral vectors for expressing sgRNA2.0, dCas9-VP64, and MS2-p65-HSF1. Each vector contains distinct selectable markers, allowing simultaneous selection of cells expressing all three vectors. b) Lentiviral delivery of SAM components was tested by first generating 293FT cell lines stably integrated with dCas9-VP64 and MS2-p65-HSF1, and then transducing these cells with single-gene-targeting lentiviral sgRNA2.0 at an MOI of <0.2. Transcriptional activation efficiency is measured 4 days after sgRNA2.0 lentiviral transduction and selection with Zeocin or puromycin. All values ​​are mean + / - SEM, n = 3. c) Flowchart of transcriptional activation screening using SAM. d) Cumulative frequency of sgRNA2.0 3 and 21 days after transduction in A375 cells. Shifts in the 21-day curve represent depletion of a subset of sgRNA2.0. Less than 0.1% of all guides are undetectable (detected by fewer than 10 reads) at day 3. e, Gene categories showing significant depletion based on Ingenuity Pathway Analysis (p<0.01 BH FDR corrected). The left category is based on the 1000 most depleted guides, and the right category is based on the 1000 genes with the greatest depletion based on the average of all three guides / genes. [Figure 69A-F]Genome-scale gene activation screening identifies mediators of BRAF inhibitor resistance. a) Box plot showing the distribution of sgRNA2.0 frequency at different time points after lentiviral transduction with or without treatment with PLX-4720. Vehicle is DMSO. Two infection replicates are shown. b) Scatter plot showing enrichment of specific sgRNA2.0 after PLX-4720 treatment. c) Identification of top candidate genes using RIGER P-value analysis (KS method) based on the average of both infection replicates. Genes are organized by location within the chromosome. d) RIGER P-values ​​for the top 100 hits from the SAM and GeCKO screens for gene perturbations that result in BRAF inhibitor resistance. e) The top 10 shared candidates from the puromycin and Zeocin screens identified using RIGER are shown. For both screens, the percentage of unique sgRNA2.0s targeting each gene present in the top 5% of all enriched sgRNA2.0s is plotted. Heatmap of fz scores. Each column represents a different BRAFV600 melanoma short-term culture. Rows represent the expression and signature of BRAF inhibitor marker genes (top panel), the expression of the top SAM screen hits (middle panel), and the screen signature score (see method for signature generation using single-sample Gene Set Enrichment Analysis) (bottom panel). Distinct transcriptional states of genes and signatures represent previously defined BRAF inhibition resistance (Konieczkowski, DJ et al. Cancer discovery 4, 816-827, doi:10.1158 / 2159-8290. CD-13-0424 (2014)). Columns are sorted by MITF expression, with highest expression indicating BRAF inhibitor sensitivity. Top hits from the SAM screen are significantly associated with resistance status (low MITF expression and high levels of resistance markers). A subset of samples was previously tested for PLX sensitivity (blue letters / arrows) and resistance (red letters / arrows). IC: Information coefficient (see Methods for details).P values ​​are generated using a permutation test (n=10,000). [Figure 70A-E] Structure-guided engineering of Cas9 sgRNAs is shown. a) Schematic of sgRNA stem loops showing contacts between each stem loop and Cas9. Contacting amino acid residues are highlighted in yellow. The tetraloop and stem-loop 2 do not have any contacts with Cas9, while stem-loops 1 and 3 share extensive contacts with Cas9. b) sgRNA2.0 with an MS2 stem-loop inserted into the tetraloop and stem-loop 2. c) Addition of a second NLS or alternative HNH domain-inactivating mutations in Cas9 improves the efficiency of transcriptional activation for MYOD1. d) The dCas9-VP64 activator shows improved performance with recruitment of MS2-P65 to the tetraloop and stem-loop 2. Addition or extension of an AU flip in the tetraloop does not increase the efficacy of dCas9-mediated transcriptional activation. e) The tetraloop and stem-loop 2 are suitable for replacement with an MS2 stem-loop. The base change from the sgRNA2.0 scaffold is indicated at each position, with the dotted line indicating the unchanged base and the base below the dotted line indicating an insertion. Deletions are indicated by the absence of the dotted line at each position. All figures are n=3 and mean ± SEM. [Figure 71A-C]Figure 2 shows that SAM mediates efficient activation of a panel of 12 genes with low levels of nonspecific activation. a) Comparison of activation levels for 12 genes using dCas9-VP64 in combination with MS2-P65, MS2-P65-HSF1, or MS2-P65-MYOD1. MS2-P65-HSF1 mediated significantly higher activation than MS2-P65 alone for 9 of the 12 genes. The best guide of the eight tested for each gene (see Figure 2a) was used in this experiment. Activation levels for each type of MS2 fusion are presented as a percentage of the activation achieved using MS2-P65. b) Nonspecific background activation by dCas9-VP64 and MS2-p65-HSF1 activator components was determined for all 12 genes. dCas9-VP64 and MS2-p65-HSF1 were cotransfected with a nontargeting (scrambled) guide. GFP-transfected cells were used to measure baseline expression levels for each gene. Only activation of IL1R2 by the scrambled guide is significantly different from the GFP sample. p<0.05 by Student's t-test. c, Mean activation for both scrambled guides shown as % of on-target activation shown in a. Activation by the scrambled guide measures less than 1% on-target activation for all 12 genes. Error bars indicate SEM. n=3. [Figure 72]Activation of characterized and uncharacterized lincRNAs by SAMs is shown. Six lincRNAs were targeted using SAMs. For each lincRNA, eight sgRNAs were designed to target the proximal promoter region (+1 to -800 bp from the TSS) using four different MS2 activators (MS2-p65-HSF1, MS2-p65-MyoD1, MS2-p65, and MS2-VP64) in combination with dCas9-VP64. The MS2 activator in combination with two different domains (MS2-p65-HSF1 or MS2-p65-MyoD1) consistently provided the greatest activation for each lincRNA, with p<0.01 for MS2-p65-HSF1 or MS2-p65-MyoD1 relative to MS2-p65. [Figure 73] Multiplexed activation using SAM is shown. Activation of a panel of 10 genes by simultaneous combinations of 2, 4, 6, or 8 sgRNAs. Average fold upregulation is shown on a log10 scale. Error bars indicate SEM, n=3. [Fig. 74A-B] Figure 1 shows activation of a panel of 12 genes as a function of SAM component dosage. a) Effect of MS2-p65-HSF1 and dCas9-VP64 dilutions, 1:1, 1:4, 1:10, and 1:50 of the original dosage of each component, on the efficacy of transcriptional upregulation. The amount of sgRNA expression plasmid is held constant. b) Effect of dilution of all three SAM components (dCas9-VP64, MS2-p65-HSF1, and sgRNA2.0) at 1:4, 1:10, and 1:50 of the original dosage for each component. Fold upregulation is calculated using GFP-transfected cells as a baseline. Error bars indicate SEM, n=3. [Fig. 75A-B]Components of cancer survival and amplification pathways are shown to be depleted in genome-wide SAM screens. IPA analysis of the top 300 depleted genes based on the average depletion of all three guides / genes resulted in two networks with scores >30. Depleted genes are shown in red. a, Network score = 39, 26 depleted genes in the network. b, Network score = 37, 25 depleted genes in the network. Components on all layers of both networks show depletion. [Fig. 76A-D] Genome-wide lentiviral screen using the puromycin-resistant SAM sgRNA2.0 library is shown. a) Cumulative frequency of sgRNA2.0 3 and 21 days after transduction of A375 cells with puromycin-resistant sgRNA2.0 lentivirus. Shifts in the 21-day curve represent depletion of a subset of sgRNA2.0. b) Box plot showing the distribution of sgRNA2.0 frequency at different time points after lentiviral transduction with the puromycin library following treatment with DMSO vehicle or PLX-4720. Two infection replicates are shown. c) Identification of top candidate genes using RIGER P-value analysis (KS method) based on the average of both infection replicates. Genes are organized by location within the chromosome. d) Overlap between the top 20 hits from the Zeo and Puro screens. Genes belonging to the same family are indicated by the same color. There is 50% overlap between the top hits from each screen, as indicated by the Venn diagram intersection. [Figure 77]Validation of the top screen hits using Cancer Cell Line Encyclopedia expression and pharmacological data from additional melanoma cell lines is shown. A heatmap of z-scores shows each column representing a different BRAFV600 melanoma cell line, and rows representing sensitivity to different drugs (top panel), expression of the SAM top screen hits (middle panel), and SAM screen signature scores (bottom panel; see signature generation method). Drug sensitivity is measured as the 8-Activity Area (AA) (Barretina, J. et al. Nature 483, 603-607, doi:10.1038 / nature11003 (2012)). Melanoma cell lines were sorted by PLX drug sensitivity, with lower values ​​(blue) corresponding to increased sensitivity. Sensitivity to relevant MAPK inhibitors is also shown. A few cell lines demonstrated resistance to MAPK inhibitors, and in these cell lines, many of the SAM top hits were highly expressed. Signatures contained within these top hits also score highly in resistant cell lines. Associations are measured using the information coefficient (IC) between PLX-4720 sensitivity (index) and each feature, with p-values ​​determined using a permutation test. RAF inhibitors: PLX4720 and RAF265; MEK inhibitors: AZD6244 and PD-0325901. [Figure 78]Validation of the top screen hits in primary and metastatic melanoma patient samples from The Cancer Genome Atlas is shown. A heatmap of z-scores shows that each column represents a different BRAFV600 patient melanoma (primary or metastatic), and rows represent the expression of gene / signature markers for BRAF inhibition sensitivity (top panel), the expression of the SAM top screen hits (middle panel), and the screen signature score (see methods for signature generation) (bottom panel). Because no pharmacological data are available for these TCGA melanoma samples, we first mapped the TCGA gene expression data onto a previously defined transcriptional state for BRAF inhibitor sensitivity / resistance based on a panel of gene markers and signatures (Konieczkowski, DJ et al. Cancer discovery 4, 816-827, doi:10.1158 / 2159-8290. CD-13-0424 (2014)). Increased expression of individual hits from the top SAM screen significantly correlates with tumors and indicates a resistant state (defined as low MITF expression and high expression of resistance markers / signatures). Signatures contained within the top genes from the SAM screen also significantly correlate with resistant tumors. A panel of melanoma samples was sorted by decreased MITF expression, with higher values ​​(red) corresponding to samples that are more sensitive to BRAF inhibition. Association is measured using the information coefficient (IC) between MITF expression (index) and each of the features, with p-values ​​determined using a permutation test. [Fig. 79A-D] Correlation between sgRNA sequence content and level of depletion of significantly depleted genes is shown. Heatmap of sgRNA nucleotide content and depletion after 21 days. sgRNAs targeting significantly depleted genes (from RIGER analysis) in the sgRNA-zeo (a, b) or sgRNA-puro (c, d) screens were analyzed for a tendency to have G content (a, c) or T content (b, d) in the sgRNA sequence. sgRNA depletion correlates positively with G content and negatively with T content. Other bases analyzed (A and C) had significant (p<0.0007) but weak (r<0.2) negative correlations. [Figure 80A-EE]1 shows an exemplary complementary sequence for Example 20. [Fig. 81A-E] 1 shows exemplary targeting guide sequences used in Example 20. [Figure 82A-I] Shown are the top 300 depleted genes for A375 in Example 20. The average depletion for each gene is listed as the log2 ratio between day 21 and day 3 averaged across all sgRNAs for that gene. [Figure 83A-C] 1 shows the top 100 genes from the output of the RIGER algorithm for the sgRNA-Zeo PLX screen comparing PLX (average of two replicates at day 21) to DMSO control (average of two replicates at day 21) in Example 20. Genes were scored using the Kolmogorov-Smirnov method. [Fig. 84A-C]

[0039] Figure 1 shows the top 100 genes from the output of the RIGER algorithm for the sgRNA-Puro PLX screen comparing PLX (average of two replicates at day 21) to DMSO control (average of two replicates at day 21) in Example 20. Genes were scored using the Kolmogorov-Smirnov method. [Figure 85] The TaqMan qPCR probe ID used to quantify relative RNA expression levels for each gene is shown (Life Technologies). [Figure 86] Crystal structure-based chimera testing between Sp, Sa, and St3Cas9 is shown. A) Domain organization and amino acid (AA) positions of SpCas9. The REC lobe is a newly identified structural component of Cas9. B) Chimera map of partial or complete swaps of the Nuc lobe with the AA positions of the chimera boundaries indicated. C) % indels achieved with each corresponding chimera on the left. Labels indicate the sgRNA used. TGS = targeting sequence (20-bp spacer portion of the sgRNA), BB = sgRNA backbone.

[0104] The figures herein are for illustrative purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION OF THE INVENTION

[0105] In particular, Applicants found that the MS2 binding loop ggccAACATGAGGATCACCCATGTCTGCAGggcc replaces nucleotides +13 to +16 and nucleotides +53 to +56 of the standard sgRNA backbone. The resulting structure is an sgRNA scaffold in which the tetraloop and stem-loop 2 sequences are replaced by the MS2 binding loop. Without being bound by theory, the tetraloop and stem-loop 2 were selected for replacement based on information obtained from the Cas9 / RNA / DNA crystal structure. Specifically, the tetraloop and stem-loop 2 were found to protrude from the Cas9 protein, suggesting that the addition of the MS2 binding loop does not interfere with any Cas9 residues. Furthermore, the proximity of the tetraloop and stem-loop 2 sites to DNA suggested that their localization results in a high degree of interaction between the DNA and any recruited proteins, such as transcriptional activators.

[0106] In some embodiments, the guide is modified such that the nucleotides corresponding to +13 to +16 and / or +53 to +56 of the standard sgRNA backbone are replaced by a discriminatory RNA.

[0107] In some embodiments, the adaptor protein is an RNA binding protein. The RNA binding protein recognizes a corresponding distinct RNA sequence, which can be an aptamer. For example, the MS2 RNA binding protein recognizes and specifically binds to the MS2 aptamer (or vice versa).

[0108] In some embodiments, the repression domain for the guide and / or CRISPR enzyme may be one shown in Example 15 to act as follows: ·SID4X domain that suppresses transcriptional activity; ·KRAB domain that suppresses transcriptional activity; NUE domains that increase repressive histone methylation; and · The NcoR domain recruits histone deacetylases that result in repressive histone modifications.

[0109] Exemplary sequences for repressor domains can be found in Example 15.

[0110] Example 15 also illustrates an orthogonal approach, see in particular Figure 48. One guide with a first aptamer / RNA binding protein pair can be bound to or fused to an activator, while a second guide with a second aptamer / RNA binding protein pair can be bound to or fused to a repressor. Because the guides are for different targets (gene loci), this allows one gene to be activated and one gene to be repressed. For example, the following scheme illustrates such an approach: Guide 1-MS2 aptamer-------MS2 RNA-binding protein-------VP64 activator; and Guide2-PP7 aptamer-------PP7 RNA binding protein-------SID4x repressor.

[0111] Figure 48 illustrates orthogonal PP7 / MS2 gene targeting. In this example, sgRNAs targeting different loci are engineered with distinct RNA loops to recruit MS2-VP64 or PP7-SID4X, which activate and repress their target loci, respectively. PP7 is an RNA-binding coat protein of the bacteriophage Pseudomonas. Like MS2, it binds to specific RNA sequences and secondary structures. The PP7 RNA recognition motif is distinct from that of MS2. Consequently, PP7 and MS2 can be multiplexed to simultaneously mediate distinct effects at different genomic loci. For example, an sgRNA targeting locus A can be engineered with an MS2 loop to recruit the MS2-VP64 activator, while another sgRNA targeting locus B can be engineered with a PP7 loop to recruit the PP7-SID4X repressor domain. Thus, dCas9 can mediate orthogonal locus-specific modifications in the same cell. This principle can be extended to incorporate other orthogonal RNA-binding proteins, such as Q-beta.

[0112] The use of two different aptamers (distinct RNAs) allows for the use of activator-adapter protein fusions and repressor-adapter protein fusions with different guides to activate expression of one gene while suppressing expression of another. These, along with their different guides, can be administered together or substantially together in a multiplexed approach. While many, e.g., 10, 20, or 30, such modified guides can all be used simultaneously, only one (or at least a minimum number) of Cas9s to be delivered can be used with a relatively small number of modified guides. The adapter protein can be associated (preferably linked or fused) with one or more activators or one or more repressors. For example, the adapter protein can be associated with a first activator and a second activator. While the first and second activators can be the same, they are preferably different activators. For example, one can be VP64 while the other can be p65, but these are merely examples and other transcriptional activators are contemplated. Three or more or even four or more activators (or repressors) can be used, but package size may limit the number, which is more than five different functional domains. A linker is preferably used for direct fusion to the adaptor protein, where two or more functional domains are associated with the adaptor protein. A suitable linker may include a GlySer linker.

[0113] It is also contemplated that the enzyme-guide complex as a whole can be associated with more than one functional domain. For example, there can be more than one functional domain associated with the enzyme, or there can be more than one functional domain associated with the guide (via one or more adaptor proteins), or there can be one or more functional domains associated with the enzyme and one or more functional domains associated with the guide (via one or more adaptor proteins).

[0114] The fusion between the adaptor protein and the activator or repressor may include a linker. For example, the GlySer linker GGGS can be used. These can be used in triple ((GGGGS)3) or 6, 9, or even 12 or more repeats to provide the appropriate length as required. Linkers can be used between the RNA binding protein and the functional domain (activator or repressor), or between the CRISPR enzyme (Cas9) and the functional domain (activator or repressor). Linkers are used to engineer the appropriate amount of "mechanical flexibility."

[0115] In some embodiments, the use of an NLS is envisaged, and applicants have found that the NLS from SV40 is useful in this regard, particularly when using lentiviral delivery methods.

[0116] In some embodiments, PP7 variants can be used. For example, applicants have found that the PP7 Pseudomonas bacteriophage coat protein (which has amino acids 68-69 mutated to SG and amino acids 70-75 deleted from the wild-type protein, as described in Wu, Bin, Jeffrey A. Chao, and Robert H. Singer. "Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells." Biophysical journal 102.12 (2012):2936-2944, and Chao, Jeffrey A., et al. "Structural basis for the coevolution of a viral RNA-protein complex." Nature structural & molecular biology 15.1 (2007):103-105) functions fully. Thus, in some embodiments, when the adaptor protein is an RNA-binding protein and the RNA-binding protein is PP7, the PP7 can be a variant as described above, i.e., a variant having amino acids 68-69 mutated to SG and / or having amino acids 70-75 deleted from the wild-type protein.

[0117] Similarly, MS2 variants can also be used, such as N55 mutants, particularly the N55K mutant, which is the N55K mutant of the MS bacteriophage coat protein (shown to have higher binding affinity than wild-type MS2 in Lim, F., M. Spingola, and D.S. Peabody. "Altering the RNA binding specificity of a translational repressor." Journal of Biological Chemistry 269, 12 (1994): 9006-9010) and was shown to be functional in Example 14.

[0118] In Example 13, Applicants showed that insertions in both the tetraloop and loop 2 were effective. In this particular example, the most efficient combination uses insertions of an aptamer (in this case, the MS2 loop; however, Applicants later show that other aptamers can also be used) in both the tetraloop and loop 2 of the sgRNA. Applicants also show that this can be used in combination with dCas9-vp64 and MS2-vp64 constructs, in other words, modifying the CRISPR enzyme as well. This new activator design (illustrated in Figure 44 and shown as red bars for the TL+L2:Ms2 guide in Figure 45) was found to mediate significantly higher target gene upregulation compared to the conventional design (illustrated in Figure 43 and shown as green bars for the generic guide in Figure 45).

[0119] It is also anticipated that other activators can be used. For example, Example 14 demonstrated an improved Cas9 activator architecture consisting of an sgRNA with a tetraloop and an MS2 loop insertion in loop 2, combined with either MS2-VP64 and dCas9-P65 or MS2-P65 and dCas9-VP64. In other words, two different activators can be used, one associated with the CRISPR enzyme (Cas9) and the other associated with the guide via an aptamer. Applicants demonstrated increased efficacy of this design compared to a standard C-terminal fusion of VP64 to Cas9. Applicants further supported the hypothesis that the combination of two different activation domains can improve target gene activation (e.g., through synergistic effects by recruiting different epigenetic modulators, general transcription factors, and coactivators). The applicants also determined that the alternative guide architecture optimized for CRISPR / Cas9 imaging in Chen, Baohui, et al. "Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR / Cas System." Cell 155.7 (2013): 1479-1491 did not offer any improvement over the standard architecture.

[0120] Of course, it is envisaged that the activator in those examples could be replaced by a repressor.

[0121] The present applicants have also investigated the placement of distinct RNA sequences (preferably aptamers) within stem-loop 2 and tetraloop of modified guides of the present invention. Example 14 further explores the use of GC tracts, which are preferred in some embodiments. GC tracts can be GC, GGGGC, CCCCG, or CGCC, or their complementary strands, or a mixture of C and G from 2 nucleotides up to, for example, 10, 15, or 20 nucleotides. In a specific example, the MS2 binding loop sequence: ggccAACATGAGGATCACCCATGTCTGCAGggcc replaced nucleotides +13 to +16 of the standard sgRNA backbone described above. Interestingly, the sequence CGCC replaced nucleotides +49 to +52 of the standard sgRNA backbone. The sequence GGCG also replaced nucleotides +57 to +60 of the standard sgRNA backbone. Tetraloop MS2 binding loop inserts were designed using the same principles as those described herein. Essentially, the CGCC and GGCG sequences replace the stem portion of stem-loop 2. It was hypothesized that the increased base pairing strength of the CGCC-GGCG stem compared to the original ACTT-AAGT stem would provide additional stability to the stem-loop 2 structure, thereby increasing sgRNA performance or longevity.

[0122] Thus, in some embodiments, one or more GC tracts may replace the stem portion of stem-loop 2. In some embodiments, one or more GC tracts may replace the stem portion of the tetraloop.

[0123] When referring to stem-loop 2 or tetraloop being modified (e.g., replaced) by a distinct RNA sequence, this preferably encompasses modification (or replacement) of the 3 or 4 nucleotides of the guide found to protrude beyond the enzyme-sgRNA-DNA complex. Suitable numbering will be evident based on the guide secondary structure itself, i.e., by searching for (or engineering) loops corresponding to stem-loop 2 and tetraloop, but exemplary numbers are around +13-16 and / or +49-52 on either side (allowing for one or two nucleotides on either side, e.g., +48-52, or +49-53, etc.).

[0124] A particularly preferred configuration is to have the aptamer followed by a GGGS linker, preferably (GGGS)3, together with an NLS, preferably an NLS from SN40.

[0125] In Example 16, Applicants generated a dCas9-based light-inducible MS2 effector characterized by an MS2-CIB1 recruitment component bound to a dCas9-sgRNA and a CRY2-VP64 transcriptional activator domain. Upon activation by blue light, CRY2-VP64 associates with MS2-CIB1, enabling recruitment of the transcription machinery to target loci.

[0126] Thus, in some embodiments, the adaptor protein can be fused to (or otherwise associated with) a first inducible element, while the functional domain can be fused to (or otherwise associated with) a second complementary inducible element. Complementarity can be provided by heterodimeric binding partners. A preferred example of the first and second complementary inducible elements is the CIB1 and CRY2 system. The CIB1 domain is a heterodimeric binding partner of light-sensitive cryptochrome 2 (CRY2).

[0127] In Example 17, Applicants replaced the dCas9Rec2 domain with a transcription effector domain; replaced the dCas9HNH domain with a transcription effector domain; inserted a transcription effector domain at the site of a flexible linker within dCas9 (amino acids 553, 575, or 1153); and created a catalytically inactive dCas9 by combining D10A and N863A mutations rather than D10A and H840A mutations, any of which may be preferred in certain distinct embodiments.

[0128] In some embodiments, Rec2 can be modified, preferably by replacing amino acids 175-306 of dCas9 with one of the following inserts, listing the subdomains from N- to C-terminus: - VP64 activation domain - 3xGGGGS linker, VP64 activation domain, 3xGGGGS linker - p65 activation domain - 3xGGGGS linker, p65 activation domain, 3xGGGGS linker

[0129] In some embodiments, the HNH can be modified. For example, the applicants replaced AA775-901 (in the HNH domain). This can be either an activator, such as vp64 or P65, or a repressor. The activator or repressor can be flanked by (GGGGS)3 or (GGGGS)6 linkers on both sides of the inserted transcription effector domain.

[0130] Insertion of transcription domains into the three loops of dCas9 is also contemplated. In addition to replacing existing domains (e.g., HNH, Rec2) with transcription effector domains, it may be useful in some embodiments to insert transcription effector domains at different positions in the Cas9 protein. Applicants identified three preferred positions: G533, F575, and K1153. The locations of G533 and K1153 in the Cas9 protein are shown in Figure 49. Applicants insert either vp64 or P65 at these three locations, flanked by (GGGGS)1 or (GGGGS)3 on both sides of the inserted transcription effector domain. Thus, in some embodiments, Cas9 can be modified by insertion of one or more functional domains at any one or more of the positions corresponding to G533, F575, and K1153 using SpCas9.

[0131] In some embodiments, novel dCas9 mutations are provided. As shown in Example 18, catalytically inactive dCas9 can be generated by combining D10A and N863A mutations rather than D10A and H840A mutations. This numbering refers to SpCas9, so the corresponding position in orthologs is assumed. Applicants also provide N580A as a preferred alternative in SaCas9, especially in combination with D10.

[0132] As shown in Example 19, N863 by SpCas9, particularly N863A, is also useful in non-functional Cas9 and is preferred in some embodiments.

[0133] This example also showed that a combination of different activator domains has an improved effect. For example, a construct with a p65-HSF1 fusion was found to be a more potent activator than a construct with p65 alone (Figures 56 and 57). Therefore, a fusion of two or more activators is preferred in some embodiments. A fusion of two or more repressors is also preferred in some embodiments. The activators or repressors can be any combination known in the art and specifically referenced herein.

[0134] Of particular note was the use in this example of an orthogonal system, a combinatorial approach of one activator and one repressor: different guides and different RNA / adapter protein pairs allowed activation at one locus and repression at another.

[0135] The applicants have observed significant activation for each of a number of difficult gene targets. Furthermore, the applicants have observed that the success rate of guide sequences typically increases with proximity to the transcription start site (TSS) of the target gene. In a preferred embodiment of the present invention, for a specific target, within 200 bp of the TSS is considered to be an advantageous width for selecting guide RNA. This information can also be useful for selecting sgRNA guide sequences.

[0136] Multiplexed activation is also demonstrated in Example 19. One important potential advantage of our system's ability to provide robust activation using a single guide is its capacity to easily activate a panel of genes simultaneously (by co-delivering multiple guides for those genes), which is challenging when multiple guides are required to activate each gene alone. To test the ability of our system (NLS-dCAS(D10,H840A)-NLS-VP64 in combination with MS2-NLS-P65-HSF1) to simultaneously activate multiple genes, we co-transfected guides targeting 2, 4, 6, 8, or 10 genes at a time. Activation of multiple genes was highly successful, and even for combinations of 10, each gene was significantly activated (see Figures 60-63). Thus, in some embodiments, the adapter protein can be advantageously fused or bound to a fusion or binding activator (further described above) or repressor, which can then be delivered to different targets by multiple guides. It is therefore particularly useful in screening methods in which the activation or repression of one or more genes is to be investigated.

[0137] Example 20 is particularly interesting. It focuses on the identification of two 4-nt stretches in the guide that are exposed on the "outside" of the Cas9-guide-target DNA complex. One 4-nt stretch fits into the tetraloop, and the other 4-nt stretch fits into stem-loop 2. These 4-nt stretches can be replaced by aptamer sequences. One or more aptamers are polynucleotides and can be DNA or RNA, although RNA is preferred. Aptamers have corresponding RNA-binding proteins that recognize specific RNA sequences.

[0138] Therefore, the MS2 system used herein includes an RNA sequence inserted into the guide (at one or both of the above locations) and the corresponding MS2 (RNA binding) protein.The RNA binding protein can then be fused to a functional domain, such as an activator or repressor.Instead of being fused directly to the functional domain, the RNA binding protein can be fused to an additional element, such as an antibody that can then bind to and recognize the functional domain, or a molecule fused to the functional domain (similar to the heteroduplex CIB1-Cry2 system described above).This can allow for greater temporal or spatial control.

[0139] In short, a specific RNA sequence can be inserted into the exposed guide loop and the corresponding RNA-binding protein can be used (whether it is fused to a functional domain or to additional elements that in turn recognize or specifically bind to the functional domain). The functional domain can be a trans-acting activator or repressor.

[0140] This can be used in screening methods to assess GOF (gain of function) and / or LOF (loss of function).

[0141] While the identification of stem-loop 2 and tetraloop has been described above, those skilled in the art may also wish to refer to Figures 44, 48, 64 (especially a and b), and 70 for guidance. Figure 70 shows the nucleotide numbering corresponding to stem-loop 2 and tetraloop. For example, in some embodiments, the tetraloop is or includes nucleotides G29 to A41 of the guide being tested, comprising 5'-GCUAGAAUAGCA-3' (positions 29-41). The guide nucleotide, e.g., C40, may preferably interact with Cas9 amino acid Arg340. In some embodiments, stem-loop 2 may be or include nucleotides A68 to G81 of the guide being used (5'-AACUUGAAAAAGUG-3'). Enzyme amino acids His1349 and Ser1351 may, in some embodiments, interact with the guide nucleotide, e.g., A68. In some embodiments, Lys33 and Tyr1356 may interact with nucleotide G81.

[0142] In some embodiments, it is preferred to use complementary GGCC inserts (GC tracts) flanking the MS insert (5'-GGCC-3' is complementary to the same sequence at the 3' end (and in the reverse orientation, i.e., 3'CCGG-5', shown in Figure 70).

[0143] While single MS2 additions (i.e., to one or the other of the tetraloop or stem-loop 2) show improvements in gain-of-function (gene upregulation) compared to standard guides, double additions (MS2 on both loops) show even stronger upregulation. Therefore, the use of two or more functional domains with guides is preferred.

[0144] As described herein, having one activator, e.g., VP64, attached to Cas9 and a separate, similar activator, also VP64, attached to the guide via MS2 in this example, shows the greatest improvement in gain-of-function (gene upregulation). Other activators or repressors can be substituted for the listed activators here.

[0145] Applicants also demonstrate in this example an improvement in terms of gain of function (gene upregulation) compared to prior art MS guide RNA configurations where MS2 is attached at the 3' end of the guide, a technology approach that differs from the loop of the present invention in that it is both internal and never at the 3' end, or at least followed (in the 3' direction) by an additional loop (stem-loop 3).

[0146] LincRNAs (non-coding RNAs produced from bidirectional promoters - the other direction is RNA corresponding to a gene of interest) can also be targeted and / or interrogated via guides.

[0147] This example also demonstrates the usefulness of lentivirus-based delivery. Overall, the system demonstrated improved transcriptional activation. Therefore, it is useful in genome-wide transcriptional activation or overexpression screening methods. For example, the present invention can be used to identify genes whose upregulation causes certain phenotypic results, in this example, resistance to BRAF kinase inhibitors in cancer cells.

[0148] Without being bound by theory, Applicants believe that guide orientation does not significantly affect activation activity; instead, the primary factor affecting activation efficacy is the location of the sgRNA site within the -200 to +1 bp proximal promoter region. Therefore, this region is the preferred target for the guide.

[0149] The adaptor protein (and thus its corresponding discriminating RNA (preferably an aptamer)) is preferably selected from among bacteriophage coat proteins. Preferred examples include those already listed elsewhere herein.

[0150] Example 21 demonstrates that an inducible structurally engineered activated mediator transgenic model (in this case, a mouse) can be established. Suppression models can also be generated similarly. Preferably, an engineered mouse is established using a Lox-Stop-polyA-Lox (LSL) cassette upstream of the coding region of the SpCas9-VP64 fusion protein. A second mouse can be engineered using a Lox-Stop-polyA-Lox (LSL) cassette upstream of the coding region of the SpCas9-VP64 fusion protein and upstream of the coding region of the MS2-P65-HSF1 fusion protein.

[0151] Example 22 investigates targeting lincRNAs of unknown function to determine abnormal phenotypes, including gain-of-function and loss-of-function studies in human cell lines (using Cre inducibility) and mice through the use of guides that target regulatory regions of genes, where the guides include activators or repressors.

[0152] When investigating lincRNAs, guides can be designed to target the promoter region. Ideally, this should be within 1000 nucleotides upstream of the target, in this case the TTS of a lincRNA of unknown function. Animals, e.g., mice, can then be screened for abnormal phenotypes.

[0153] Cells with activator sgRNAs can be monitored for gain of function, while cells with repressor sgRNAs can be monitored for loss of function. In this manner, mammalian, e.g., mouse and human, cells can be screened.

[0154] In one aspect, the vector system used in the methods of the present invention comprises one or more lentiviral vectors. In a preferred embodiment, the one or more lentiviral vectors may comprise a codon-optimized nuclear localization signal (NLS), a codon-optimized P2A bicistronic linker sequence, and an optimally positioned U6 drive guide RNA cassette. In another aspect, the vector system comprises two lentiviral vectors, one of which comprises a Cas9 enzyme and the other of which comprises a guide RNA selected from the library of the present invention. In one embodiment of the present invention, each vector has a different selectable marker, for example, a different antibiotic resistance marker. The present invention also encompasses kits comprising the library of the present invention. In one aspect, the kit comprises a single container containing a vector comprising the library of the present invention. In another aspect, the kit comprises a single container containing a plasmid comprising the library of the present invention. The present invention also encompasses kits comprising a panel comprising a selection of unique CRISPR-Cas system guide sequences from the library of the present invention, wherein the selection indicates a specific physiological state. In a preferred embodiment, the targeting is greater than about 100 sequences, greater than about 1,000 sequences, or greater than about 20,000 sequences, or the entire genome. In other embodiments, the panel of target sequences is focused on relevant or desired pathways, such as immune pathways or cell division.

[0155] Thus, Examples 21 and 22 demonstrate that it is feasible to provide for the generation of non-human animals or cells. These are preferably modified to constitutively or conditionally express Cas9 with one or more mutations that alter catalytic activity as described herein, or are the descendants of such modified animals or cells. The models can be used for screening using appropriate guides and the activators or repressors described herein for multiplexing to show up- and / or down-regulation of different adaptor and target gene functions. Corresponding cell lines and transgenic mammalian models are therefore provided. Further guidance regarding models and cell lines is provided herein.

[0156] The exposed or external portion of the guide (when the guide-Cas9-DNA complex is formed) is preferably a 4-nucleotide stretch. In some embodiments, the stretch may be in a tetraloop. In some embodiments, the stretch may be in stem-loop 2. In some embodiments, stretches in both the tetraloop and stem-loop 2 are envisioned.

[0157] This stretch can be modified, altered, or completely replaced. Generally, reducing the number of nucleotides in the exposed stretch to less than four is not preferred for steric reasons, as this may affect the secondary structure of the remainder of the guide and thus affect the formation of the Cas9-guide-DNA complex or the exposure of the stretch.

[0158] This can be modified or changed so that all four of the original tetranucleotides in the stretch are retained, with additions (or further nucleotides) being made between 1 and 2, between 2 and 3, or between 3 and 4. It is also envisaged that additions can be made directly 5' to 1, or 3' to 4. The stem can be flexible, but is preferably largely self-complementary overall.

[0159] Unafold is a software tool that can be used to aid in guide RNA secondary structure prediction and thus assist those skilled in the art in determining which changes in a guide RNA may be tolerable within the framework described herein.

[0160] Ideally, loop features should be maintained, but this is determined by the protein-binding section of the differentiated RNA that is added to the guide.The non-loop ends adjacent to the end of the enzyme should ideally be maintained in the sense that they must be present, but the primary sequence of the original guide can be changed, for example, by inserting one or more GC tracts.Ideally, this should be done in the non-loop (non-protein-binding end) of the differentiated RNA that is added, which can be extended.The secondary structure of the non-protein-binding region of the differentiated RNA should preferably form a stem.

[0161] It is preferable to avoid bulges or loops in the exposed section (the non-protein-binding section of the distinguishing RNA, i.e., between the end of the enzyme complex and the protein-binding domain of the distinguishing RNA / aptamer). Rather, it is preferable to retain the stem as the secondary structure of the exposed section.

[0162] Stems can be formed in RNA through the use of complementary sections of approximately equal length to minimize mismatches. The maximum length of the stem (or the number of nucleotides forming the stem in both the 5' to 3' and 3' to 5' strands) is preferably 100 nucleotides, or a total of 100 nucleotides (i.e., two sections of approximately 50 nucleotides each) to reduce steric effects and the potential formation of secondary or tertiary structures with additional nucleotides. However, while 50 to 60 nucleotides may be a more preferred maximum, given the general need to maintain reduced package size, 10 to 20 or 30 nucleotides are most preferred, while 8, 10, or 12 nucleotides are most preferred.

[0163] A preferred minimum length is 4 nucleotides on either side of the protein binding loop.

[0164] Also provided is a method for upregulating gene expression in a target locus, comprising administering a modified guide of the present invention directed to the target, wherein the adaptor protein is associated with an activator. CRISPR enzymes can also be modified with functional domains.

[0165] Also provided is a method for downregulating gene expression in a target locus, comprising administering a modified guide of the present invention directed to the target, wherein the adaptor protein is associated with a repressor. CRISPR enzymes can also be modified with functional domains.

[0166] Such methods can be suitably used in methods of treating a subject in need of treatment, e.g., a subject requiring gene upregulation or gene downregulation. For example, by following the orthogonal approach described herein, multiplex methods can be used to upregulate some genes and downregulate others.

[0167] Also provided are compositions and systems of the invention for use in such methods of treatment. Also provided is the use of compositions and systems of the invention in the manufacture of a medicament for such treatment.

[0168] With respect to guides generally, and specifically to guides for modified sgRNAs of the present invention and complexes formed therewith, it is preferred that the guide have one or more of the following characteristics: In some embodiments, the tracr sequence has one or more hairpins and is 30 nucleotides or more in length, more preferably 40 nucleotides or more in length, or more preferably 50 nucleotides or more in length. In some embodiments, the guide sequence is 10 to 30 nucleotides in length. In some embodiments, the CRISPR / Cas enzyme is a type II Cas9 enzyme. In some embodiments, the tracr sequence has one or more hairpins and is 30 nucleotides or more in length, more preferably 40 nucleotides or more in length, or more preferably 50 nucleotides or more in length, the guide sequence is 10 to 30 nucleotides in length, and the CRISPR / Cas enzyme is a type II Cas9 enzyme.

[0169] Endogenous transcriptional repression is often mediated by chromatin-modifying enzymes, such as histone methyltransferases (HMTs) and deacetylases (HDACs). Repressive histone effector domains are known, and an exemplary list is provided below. In the exemplary table, small protein sizes and functional truncations are preferred to facilitate efficient viral packaging (e.g., via AAV). However, in general, domains can include HDAC, histone methyltransferase (HMT), and histone acetyltransferase (HAT) inhibitors, as well as HDAC and HMT recruiting proteins. In some embodiments, functional domains can be or include HDAC effector domains, HDAC recruiter effector domains, histone methyltransferase (HMT) effector domains, histone methyltransferase (HMT) recruiter effector domains, or histone acetyltransferase inhibitor effector domains.

[0170] [Table 1]

[0171] Thus, the repressor domain of the present invention can be selected from histone methyltransferase (HMT), histone deacetylase (HDAC), histone acetyltransferase (HAT) inhibitors, and HDAC and HMT recruiting proteins.

[0172] The HDAC domain can be any of those in the table above: HDAC8, RPD3, MesoLo4, HDAC11, HDT1, SIRT3, HST2, CobB, HST2, SIRT5, Sir2A, or SIRT6.

[0173] In some embodiments, the functional domain may be an HDAC recruiter effector domain. Preferred examples include those in the following table: MeCP2, MBD2b, Sin3a, NcoR, SALL1, and RCOR1. While NcoR is exemplified and preferred in this example, it is envisioned that others in this class will also be useful.

[0174] [Table 2]

[0175] In some embodiments, the functional domain can be a methyltransferase (HMT) effector domain. Preferred examples include those in the table below: NUE, vSET, EHMT2 / G9A, SUV39H1, dim-5, KYP, SUVR4, SET4, SET1, SETD8, and TgSET8. While NUE is exemplified in this example and is preferred, it is envisioned that others in this class will also be useful.

[0176] [Table 3]

[0177] In some embodiments, the functional domain may be a histone methyltransferase (HMT) recruiter effector domain. Preferred examples include those in the table below: Hp1a, PHF19, and NIPP1.

[0178] [Table 4]

[0179] In some embodiments, the functional domain may be a histone acetyltransferase inhibitor effector domain. Preferred examples include SET / TAF-1β, listed in the table below.

[0180] [Table 5]

[0181] In addition to promoters or promoter-proximal elements, it is also preferable to target endogenous (regulatory) control elements (for example, enhancers and silencers).Therefore, in addition to targeting promoters, the present invention can also be used to target endogenous control elements (for example, enhancers and silencers).These control elements can be located upstream and downstream of the transcription start site (TSS), starting from 200 bp from the TSS and 100 kb apart.Targeting known control elements can be used to activate or repress target genes.In some cases, a single control element can affect the transcription of multiple target genes.Therefore, targeting a single control element can be used to simultaneously control the transcription of multiple genes.

[0182] On the other hand, targeting putative regulatory elements (e.g., by tiling the region of the putative regulatory element and 200 bp to up to 100 kb surrounding the element) can be used as a means to confirm such elements (by measuring transcription of the gene of interest) or to detect novel regulatory elements (e.g., by tiling 100 kb upstream and downstream of the TSS of the gene of interest). Furthermore, targeting putative regulatory elements can be useful for understanding the genetic causes of disease. Many mutations and common SNP variants associated with disease phenotypes are located outside of coding regions. Following targeting of such regions with either the activation or repression systems described herein, transcriptional readouts can be obtained, either a) a set of putative targets (e.g., a set of genes located in close proximity to the regulatory element) or b) a whole transcriptome readout, e.g., by RNA sequencing or microarray. This allows for the identification of candidate genes likely involved in the disease phenotype. Such candidate genes can be useful as novel drug targets.

[0183] The term "associated with" is used herein to refer to how one molecule "associates" with another molecule, for example, between an adaptor protein and a functional domain, or between a CRISPR enzyme and a functional domain. In the case of such protein-protein interactions, this association can be understood in terms of recognition, such as an antibody recognizing an epitope. Alternatively, a protein can be associated with another protein through a fusion of the two, for example, one subunit fused to another subunit. Fusion typically occurs by adding one amino acid sequence to the other, for example, by splicing together the nucleotide sequences encoding the respective proteins or subunits. Alternatively, this can essentially be understood as a bond or direct bond between two molecules, for example, a fusion protein. In any event, the fusion protein can include a linker between the two subunits of interest (i.e., between the enzyme and the functional domain or between the adaptor protein and the functional domain). Thus, in some embodiments, a CRISPR enzyme or adaptor protein is associated with a functional domain by binding to it. In other embodiments, the CRISPR enzyme or adaptor protein is associated with the functional domain because the two are fused together, optionally via an intermediate linker.

[0184] RNA sequences that bind to protein sequences, particularly aptamers, are known, but the way they bind to, for example, adaptor proteins is that RNA sequences recognize the corresponding RNA binding domain or portion on protein and form a complex with it.This is similar to the way that antibodies recognize epitopes.Therefore, in some embodiments, distinct RNA sequences recognize and bind to the complementary RNA binding domain or portion on adaptor proteins.In some embodiments, distinct RNA sequences are aptamers.It is well known that the function of aptamers is to associate with their corresponding proteins.

[0185] A distinct RNA sequence is a sequence that differs in origin and / or sequence from the guide into which it is inserted. The insertion may involve one or more substitutions (deletions) of the original guide nucleotides at the insertion site. Alternatively, the original guide nucleotides can be retained by the insertion site between the inserted nucleotides, such that the inserted nucleotides separate the adjacent (primary structure) original nucleotides. Thus, a distinct RNA sequence may differ in the sense that it has a different primary structure (nucleotide sequence) from the nucleotides it replaces. In either case, in the case of substitution or simple insertion without deletion, the overall primary sequence of the resulting modified guide will change. Thus, in one embodiment, a distinct RNA sequence is one that results in a different sequence (primary structure) in the resulting modified guide.

[0186] In some embodiments, the methods provided herein can be performed ex vivo, unless otherwise specified.

[0187] In both Example 9 and Example 24, the applicants found that the helical domain 2 (HD2) of SpCas9 can be deleted. While some activity may be lost, this may only be about 50%; this truncation may be advantageous. Although relatively small, a moderate reduction in the total number of amino acids for SpCas9 is observed. This may only be useful for encoding Cas9 and a guide into a single vector for packaging or delivery of Cas9. In some situations, a functional but less active Cas9, or a relatively active Cas9, may be advantageous; for example, when off-target effects are a concern or when a functional domain is associated with a CRISPR Cas9 complex (e.g., with a Cas9 protein). In place of the truncated portion of Cas9, the applicants added a linker in Example 9.

[0188] Applicants also found in Example 24 that chimeric three-component enzymes can be created that focus on the HD2 and surrounding regions of SpCas9. To date, research has focused on creating chimeras in which the N'- or C'-terminal domains have been swapped out from SpCas9 and replaced with the corresponding domains from StCas9, e.g., providing [N'-terminal SpCas9-C'-terminal StCas9] chimeras. While these two-component chimeras are useful, Applicants have now found that chimeric three-component enzymes are possible and functional.

[0189] Thus, in one aspect, the present invention provides a CRISPR enzyme or Cas enzyme, preferably Cas9, in which the HD2 domain has been truncated. In some embodiments, the truncation is a replacement of the HD2 domain (i.e., in its entirety). In some embodiments, the truncation is a replacement of the HD2 domain, or a truncated portion thereof, with a linker, preferably GlySer or other flexible linker, or a rigid linker, such as an alpha-helical linker (Ala(GluAlaAlaAlaLys)Ala). A suitable example of an HD2 domain (in this example, from SpCas9) is provided in Example 9, and examples of truncated sequences lacking the HD2 domain are also provided for guidance.

[0190] In some embodiments, the CRISPR enzyme is selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphingobacterium, Sphingobacterium sp., Sphingobacterium spp ... Cas9 orthologs from genera belonging to the group consisting of Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, and Campylobacter, where Cas contains a helical domain 2 truncation.

[0191] In some embodiments, to replace helical domain 2, the helical domain 2 truncation is replaced with one or more sets of flexible glycine-serine (GlyGlyGlySer) or rigid alpha-helical linkers (Ala(GluAlaAlaAlaLys)Ala) in groups of 1, 3, 6, 9, or 12 repeats, which may provide potential structural stabilization and / or aid in retaining Cas9:sgRNA specificity.

[0192] Therefore, in another aspect, the present invention provides a chimeric three-component CRISPR enzyme or Cas enzyme, preferably Cas9. The chimeric three-component enzyme preferably comprises the N'- and C'-terminal components from a first CRISPR enzyme or Cas enzyme, such as SpCas9, and an internal component from a different enzyme, i.e., a second CRISPR enzyme or Cas enzyme. The second CRISPR enzyme or Cas enzyme is typically an orthologue of the first CRISPR enzyme or Cas enzyme. In some embodiments, the second CRISPR enzyme or Cas enzyme can be any of the orthologues described herein. In some embodiments, the second CRISPR enzyme or Cas enzyme is Sa. In some embodiments, the second CRISPR enzyme or Cas enzyme is St enzyme. In some embodiments, the second CRISPR enzyme or Cas enzyme is St3 enzyme.

[0193] The internal components of the second CRISPR or Cas enzyme do not include any amino acids at or within 2 or 3 amino acids from the N'- or C'-terminal end of the functional wild-type second CRISPR or Cas enzyme (including any post-translational processing). The minimum distance from the N'- or C'-terminal end of the functional wild-type second CRISPR or Cas enzyme is ideally 5 to 10 amino acids in some embodiments.

[0194] In some embodiments, the components of the first CRISPR or Cas enzyme or the second CRISPR or Cas enzyme may comprise at least one domain or span the boundary between two or more domains. An example of this can be found in Example 24 and Figure 86B, where the components swapped into and out of SpCas9 (the first CRISPR or Cas enzyme) span the boundary between the various domains identified in Figure 86A. In some embodiments, one or more complete domains are preferred. In some embodiments, one or more partial domains are preferred. In some embodiments, the Rec lobe is completely or partially swapped out, so that the internal components from the second CRISPR or Cas enzyme contain a complete or partial Rec lobe. Thus, the N'- and C'-terminal components from the first CRISPR or Cas enzyme lack the Rec lobe.

[0195] In some embodiments, the internal component of the second CRISPR enzyme or Cas enzyme is an HD2 domain.

[0196] In some embodiments, internal components of the second CRISPR or Cas enzyme replace one or more corresponding components in the first CRISPR or Cas enzyme. In some embodiments, the HD2 domain of the first CRISPR or Cas enzyme is completely truncated or replaced. Truncation can be by at least 20%, more preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%, more preferably by at least 60%, more preferably by at least 70%, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, more preferably by at least 95%, more preferably by at least 98%, and more preferably by at least 99%. As mentioned above, a suitable example of an HD2 domain (in this case, from SpCas9) is provided in Example 9, and examples of truncated sequences lacking the HD2 domain are also provided for guidance. In some embodiments, a moiety can be the only part of a domain, for example, a preferred N'-terminal moiety is amino acids 1-10 of Sp, which is the only part of the RuvCI domain in Sp.

[0197] In some embodiments, an internal component from a first CRISPR or Cas enzyme is replaced by an internal component from a second CRISPR or Cas enzyme, and the two internal components can be identical (i.e., correspond to each other between orthologs, for example, comparable by sequence alignment) or different.

[0198] In some embodiments, the chimeric three-component enzyme comprises the N'- and C'-terminal components from SpCas9 and the internal domain from Sa or St3, providing an Sp-St3-Sp or Sp-Sa-Sp chimeric three-component enzyme (N'-to-C' orientation).

[0199] In some embodiments, the N'-terminal component of the first CRISPR or Cas enzyme is Sp1-10 (amino acids 1-10 of SpCas9). In some embodiments, the N'-terminal component of the first CRISPR or Cas enzyme is Sp1-189. In some embodiments, the N'-terminal component of the first CRISPR or Cas enzyme is SP1-299.

[0200] In some embodiments, the C'-terminal component of the first CRISPR or Cas enzyme is SP729-1404. In some embodiments, the C'-terminal component of the first CRISPR or Cas enzyme is Sp190-1404. In some embodiments, the C'-terminal component of the first CRISPR or Cas enzyme is Sp328-1404.

[0201] In some embodiments, the internal component of the second CRISPR enzyme or Cas enzyme is St3 87-712. In some embodiments, the internal component of the second CRISPR enzyme or Cas enzyme is St3 174-712. In some embodiments, the internal component of the second CRISPR enzyme or Cas enzyme is St3 87-173. In some embodiments, the internal component of the second CRISPR enzyme or Cas enzyme is St3 174-311. In some embodiments, the internal component of the second CRISPR enzyme or Cas enzyme is St3 312-712. Thus, in some embodiments, a suitable lower limit for the internal component of the second CRISPR enzyme or Cas enzyme is St3 87, or 174, or 312. Furthermore, in some embodiments, a suitable upper limit for the internal component of the second CRISPR enzyme or Cas enzyme is St3 712, or 172 / 173, or 311. Any of these combinations is preferred, except that the upper and lower limit points are adjacent to or within at least 10 amino acids of each other.

[0202] In some embodiments, the internal component of the second CRISPR or Cas enzyme is Sa125-200. In some embodiments, the internal component of the second CRISPR or Cas enzyme is Sa125-200, the N'-terminal component of the first CRISPR or Cas enzyme is Sp1-189, and the C'-terminal component of the first CRISPR or Cas enzyme is Sp328-1404.

[0203] Some variation of these boundaries (except where the amino acid is the very first or very last in the wild type) is envisioned, keeping in mind the above requirements regarding distance from the ends. Preferred ranges for these boundaries are within a region of 1, 2, 3, 4, 5, 8, 10, 15, or 20 amino acids.

[0204] It is recognized that a chimeric three-component CRISPR enzyme or Cas enzyme is functional and preferably has at least at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99% or more preferably 100% or more of the activity of a wild-type enzyme (an enzyme that has not been modified as described herein).

[0205] In some embodiments, hybrid guides are preferred for use with chimeric three-component CRISPR enzymes or Cas9 enzymes. The hybrid guide comprises a backbone and a targeting sequence. The backbone comprises a tracrRNA scaffold (tracr mate and tracr sequence), and the targeting sequence comprises an approximately 20nt guide (spacer) sequence for DNA targeting. The backbone can correspond to that of an endogenous guide from the same species as the first CRISPR enzyme or Cas enzyme, or the backbone can correspond to that of an endogenous guide from the same species as the second CRISPR enzyme or Cas enzyme. The targeting sequence can correspond to that of an endogenous guide from the same species as the first CRISPR enzyme or Cas enzyme, or the targeting sequence can correspond to that of an endogenous guide from the same species as the second CRISPR enzyme or Cas enzyme.

[0206] Exemplary configurations for truncated and chimeric three-component CRISPR or Cas9 enzymes are shown in Figures 27A and B with reference to wtSpCas9, except that the last chimera in Figure 27B is a two-component chimera. The truncation shown in the third configuration in Figure 27A is preferred because it is an HD2 truncation in which the HDS region is replaced by a linker. A suitable linker is the GlySer linker or alpha-helical linker (Ala(GluAlaAlaAlaLys)Ala) described herein.

[0207] The CRISPR-Cas enzyme described herein is preferably a type II CRISPR-Cas enzyme.In some embodiments, in the CRISPR complex, the tracr sequence has one or more hairpins and is 30 nucleotides or more, 40 nucleotides or more, or 50 nucleotides or more; the guide sequence is 10 to 30 nucleotides in length, and the CRISPR / Cas enzyme may preferably be a type II Cas9 enzyme.

[0208] For general information regarding CRISPR-Cas systems, their components, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, AAV, and their production and use, including amounts and formulations, all useful in practicing the present invention, reference is made to the following: U.S. Patent Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,889,418, and 8,895,308;U.S. Patent Application Publication No. 2014-0310830 (U.S. Patent Application No. 14 / 105,031), U.S. Patent Application No. 2014-0287938A1 (U.S. Patent Application No. 14 / 213,991), U.S. Patent Application No. 2014-0273234A1 (U.S. Patent Application No. 14 / 293,674), U.S. Patent Application No. 2014-0273232A1 (U.S. Patent Application No. 14 / 290,575), U.S. Patent Application No. 2014-0273231 (U.S. Patent Application No. 14 / 259,420), U.S. Patent Application No. 2014-0256 No. 046A1 (U.S. Patent Application No. 14 / 226,274), No. 2014-0248702A1 (U.S. Patent Application No. 14 / 258,458), No. 2014-0242700A1 (U.S. Patent Application No. 14 / 222,930), No. 2014-0242699A1 (U.S. Patent Application No. 14 / 183,512), No. 2014-0242664A1 (U.S. Patent Application No. 14 / 104,990), No. 2014-0234972A1 (U.S. Patent No. 14 / 183,471), No. 2014-0227787A1 (U.S. Patent Application No. 14 / 256,912), No. 2014-0189896A1 (U.S. Patent Application No. 14 / 105,035), No. 2014-0186958 (U.S. Patent Application No. 14 / 105,017), No. 2014-0186919A1 (U.S. Patent Application No. 14 / 104,977), No. 2014-0186843A1 (U.S. Patent Application No. 14 / 104,900 Nos. 2014-0179770A1 (U.S. Patent Application Nos. 14 / 104,837) and 2014-0179006A1 (U.S. Patent Application Nos. 14 / 183,486), 2014-0170753 (U.S. Patent Application No. 14 / 183,429); European Patent Application Nos. 2771468 (EP13818570.7), 2764103 (EP13824232.6), and 2784162 (EP14170383.5);and PCT patent application publications WO 2014 / 093661 (PCT / US2013 / 074743), WO 2014 / 093694 (PCT / US2013 / 074790), WO 2014 / 093595 (PCT / US2013 / 074611), WO 2014 / 093718 (PCT / US2013 / 074825), WO 2014 / 093709 (PCT / US2013 / 074812), WO 2014 / 093622 (PCT / US2013 / 074667), WO 2014 / 093635 (PCT / US2013 / 074691), WO 2014 / 093655 (PCT / US2013 / 074736), WO 2014 / 093712 (PCT / US2013 / 074819), WO 2014 / 093701 (PCT / US2013 / 074800), and WO 2014 / 018423 (PCT / US2013 / 051418). 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PCT / US2014 / 041803, PCT / US2014 / 041800, PCT / US2014 / 041809, PCT / US2014 / 041804, and PCT / US2014 / 041806, each filed June 10, 2014; PCT / US2014 / 041808, filed June 11, 2014; and PCT / US2014 / 041809, filed June 12, 2014. No. PCT / US2014 / 62558, filed October 28, 2014, and U.S. Provisional Patent Applications Nos. 61 / 915,150, 61 / 915,301, 61 / 915,267, and 61 / 915,260, filed December 12, 2013, respectively; Nos. 61 / 757,972 and 61 / 768,959, filed January 29, 2013 and February 25, 2013; and Nos. 61 / 757,972 and 61 / 768,959, filed June 17, 2013. Nos. 61 / 835,936, 61 / 836,127, 61 / 836,101, 61 / 836,080, 61 / 835,973, and 61 / 835,931, both filed June 11, 2014; Nos. 62 / 010,888 and 62 / 010,879, both filed June 11, 2014; and Nos. 62 / 010,329 and 62 / 010,441, each filed June 10, 2014. Nos. 61 / 939,228 and 61 / 939,242, each filed February 12, 2014; No. 61 / 980,012, filed April 15, 2014; No. 62 / 038,358, filed August 17, 2014; Nos. 62 / 054,490, 62 / 055,484, 62 / 055,460, and 62 / 055,487, each filed September 25, 2014;and U.S. Provisional Patent Application Nos. 62 / 069,243, filed October 27, 2014. Also reference is made to U.S. Provisional Patent Applications Nos. 62 / 055,484, 62 / 055,460, and 62 / 055,487, filed September 25, 2014; U.S. Provisional Patent Application No. 61 / 980,012, filed April 15, 2014; and U.S. Provisional Patent Application No. 61 / 939,242, filed February 12, 2014. Reference is made to PCT applications, particularly U.S. Designated Application No. PCT / US14 / 41806, filed June 10, 2014. Reference is made to U.S. Provisional Patent Application No. 61 / 930,214, filed January 22, 2014. Reference is made to U.S. Provisional Patent Applications Nos. 61 / 915,251; 61 / 915,260, and 61 / 915,267, each filed December 12, 2013. Reference is made to U.S. Provisional Patent Application No. USSN 61 / 980,012, filed April 15, 2014. Reference is made to PCT applications, particularly U.S. Designated Application No. PCT / US14 / 41806, filed June 10, 2014. Reference is made to U.S. Provisional Patent Application No. 61 / 930,214, filed January 22, 2014. Reference is made to U.S. Provisional Patent Applications Nos. 61 / 915,251; 61 / 915,260, and 61 / 915,267, each filed December 12, 2013. Each of these patents, patent application publications, and applications, and all documents cited in those applications or during their prosecution ("application references") and all documents cited or referenced in those application references, together with any manufacturer's instructions, manuals, product specifications, and product sheets for any products mentioned in those documents or in any documents therein incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of this invention. All documents (e.g., these patents, patent application publications, and applications and application references) are incorporated by reference herein to the same extent as if each individual document were individually and specifically indicated to be incorporated by reference;

[0209] Further, for general information regarding CRISPR-Cas systems, the following may be mentioned (further incorporated herein by reference): - Multiplex genome engineering using CRISPR / Cas systems.Cong,L.,Ran,FA,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,PD,Wu,X.,Jiang,W.,Marraffini,LA,&Zhang,F.Science Feb 15;339(6121):819-23(2013); - RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Jiang W.,Bikard D.,Cox D.,Zhang F,Marraffini LA.Nat Biotechnol Mar;31(3):233-9(2013); - One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR / Cas-Mediated Genome Engineering. Wang H., Yang H., Shivaila CS., Dawlaty MM., Cheng AW., Zhang F., Jaenisch R. Cell May 9;153(4):910-8(2013); - Optical control of mammalian endogenous transcription and epigenetic states.Konermann S,Brigham MD,Trevino AE,Hsu PD,Heidenreich M,Cong L,Platt RJ,Scott DA,Church GM,Zhang F.Nature.2013 Aug 22;500(7463):472-6.doi:10.1038 / Nature12466.Epub 2013 Aug 23; - Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity.Ran,FA.,Hsu,PD.,Lin,CY.,Gootenberg,JS.,Konermann,S.,Trevino,AE.,Scott,DA.,Inoue,A.,Matoba,S.,Zhang,Y.,&Zhang,F.Cell Aug 28.pii:S0092-8674(13)01015-5.(2013); - DNA targeting specificity of RNA-guided Cas9 nucleases.Hsu,P.,Scott,D.,Weinstein,J.,Ran,FA.,Konermann,S.,Agarwala,V.,Li,Y.,Fine,E.,Wu,X.,Shalem,O.,Cradick,TJ.,Marraffini,LA.,Bao,G.,&Zhang,F.Nat Biotechnol doi:10.1038 / nbt.2647(2013); - Genome engineering using the CRISPR-Cas9 system.Ran,FA.,Hsu,PD.,Wright,J.,Agarwala,V.,Scott,DA.,Zhang,F.Nature Protocols Nov;8(11):2281-308.(2013); - Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.Shalem,O.,Sanjana,NE.,Hartenian,E.,Shi,X.,Scott,DA.,Mikkelson,T.,Heckl,D.,Ebert,BL.,Root,DE.,Doench,JG.,Zhang,F.Science Dec 12.(2013).[Epub ahead of print]; - Crystal structure of cas9 in complex with guide RNA and target DNA.Nishimasu,H.,Ran,FA.,Hsu,PD.,Konermann,S.,Shehata,SI.,Dohmae,N.,Ishitani,R.,Zhang,F.,Nureki,O.Cell Feb 27.(2014).156(5):935-49; - Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.Wu X.,Scott DA.,Kriz AJ.,Chiu AC.,Hsu PD.,Dadon DB.,Cheng AW.,Trevino AE.,Konermann S.,Chen S.,Jaenisch R.,Zhang F.,Sharp PA.Nat Biotechnol.(2014)Apr 20.doi:10.1038 / nbt.2889、 - CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling,Platt et al.,Cell 159(2):440-455(2014)DOI:10.1016 / j.cell.2014.09.014、 - Development and Applications of CRISPR-Cas9 for Genome Engineering,Hsu et al,Cell 157,1262-1278(June 5,2014)(Hsu 2014)、 - Genetic screens in human cells using the CRISPR / Cas9 system,Wang et al.,Science.2014 January 3;343(6166):80-84.doi:10.1126 / science.1246981、 - Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation, Doench et al., Nature Biotechnology published online 3 September 2014;doi:10.1038 / nbt.3026, and - In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9, Swiech et al, Nature Biotechnology; published online 19 October 2014; doi:10.1038 / nbt.3055 (each of which is incorporated herein by reference and described briefly below).

[0210] Cong et al. engineered a type II CRISPR / Cas system for use in eukaryotic cells based on both Streptococcus thermophilus Cas9 and Streptococcus pyogenes Cas9, demonstrating that Cas9 nuclease can be directed by short RNAs to induce precise cleavage of DNA in human and mouse cells. Their work further demonstrated that Cas9 converted into a nicking enzyme can be used to facilitate homologous recombination repair in eukaryotic cells with minimal mutagenic activity. Furthermore, their work demonstrated that multiple guide sequences can be encoded in a single CRISPR array to enable simultaneous editing of several endogenous genomic loci within mammalian genomes, demonstrating the easy programmability and broad applicability of RNA-guided nuclease technology. This ability to use RNA to program sequence-specific DNA cleavage in cells defines a new class of genome engineering tool. The authors' work further demonstrated that other CRISPR loci are likely to be transplantable into mammalian cells and may also mediate mammalian genome cleavage. Importantly, it is envisioned that some aspects of the CRISPR / Cas system can be further improved to increase its efficiency and versatility.

[0211] Jiang et al. used clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed with duplex RNA to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. This approach relies on duplex RNA:Cas9-directed cleavage at targeted genomic sites to kill non-mutated cells, circumventing the need for selectable markers or counterselection systems. This study reported reprogramming of duplex RNA:Cas9 specificity by altering the sequence of a short CRISPR RNA (crRNA) to generate single- and multi-nucleotide changes carried on the editing template. This study demonstrated that the simultaneous use of two crRNAs enables multiplex mutagenesis. Furthermore, when this approach was used in combination with recombineering, in S. pneumoniae, nearly 100% of the cells recovered using the described approach contained the desired mutation, and in E. coli, 65% of the recoveries contained the mutation.

[0212] Konermann et al. addressed the need in the art for a versatile and robust technology that allows for optical and chemical modulation of DNA-binding domain-based CRISPR Cas9 enzymes and also transcription activator-like effectors.

[0213] The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20-nt guide sequence, which can tolerate some mismatches in the DNA target and thereby promote undesired off-target mutagenesis. To address this, Ran et al. described an approach in which a Cas9 nickase mutant is combined with a paired guide RNA to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, double-strand breaks require simultaneous nicking via appropriately spaced guide RNAs, extending the number of bases specifically recognized for target cleavage. The authors demonstrated that the use of paired nicking reduced off-target activity by 50- to 1,500-fold in cell lines, facilitating gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide range of genome editing applications requiring high specificity.

[0214] Hsu et al. characterized SpCas9 targeting specificity in human cells to provide target site selection and avoid off-target effects. This study evaluated SpCas9-induced indel mutation levels at >700 guide RNA variants and >100 predicted genomic off-target loci in 293T and 293FT cells. The authors reported that SpCas9 tolerates mismatches between guide RNA and target RNA at different positions in a sequence-dependent manner that is sensitive to the number, position, and distribution of mismatches. They further demonstrated that SpCas9-mediated cleavage is not affected by DNA methylation and that titration of SpCas9 and sgRNA dosage minimizes off-target modifications. Furthermore, to facilitate mammalian genome engineering applications, the authors reported the provision of web-based software tools to guide target sequence selection and validation and off-target analysis.

[0215] Ran et al. described a set of tools for Cas9-mediated genome editing via non-homologous end joining (NHEJ) or homology-directed repair (HDR) in mammals, as well as the generation of engineered cell lines for downstream functional studies. To minimize off-target cleavage, the authors further described a double-nicking strategy using paired guide RNAs and a Cas9 nickase mutant. The protocol provided by the authors experimentally guided guidelines for target site selection, evaluation of cleavage efficiency, and analysis of off-target activity. This study demonstrated that, starting from target design, genetic modification can be achieved as quickly as within 1–2 weeks, and engineered clonal cell lines can be derived within 2–3 weeks.

[0216] Shalem et al. described a novel approach to interrogate gene function on a genome-wide scale. Their study demonstrated that delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted 18,080 genes, with 64,751 unique guide sequences enabling both negative and positive selection screening in human cells. First, the authors demonstrated the use of the GeCKO library to identify genes essential for cell survival in cancer and pluripotent stem cells. Next, in a melanoma model, the authors screened for genes whose loss contributes to resistance to vemurafenib, a therapeutic agent that inhibits the mutant protein kinase BRAF. Their study showed that the top-ranked candidates included the previously validated genes NF1 and MED12 as well as novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a high level of concordance and a high rate of hit confirmation between independent guide RNAs targeting the same gene, thus demonstrating the promise of genome-scale screening using Cas9.

[0217] Nishimasu et al. reported the crystal structure of Streptococcus pyogenes Cas9 in complex with an sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of a target recognition and nuclease lobe that accommodates the sgRNA:DNA heteroduplex in a positively charged groove at the interface. While the recognition lobe is essential for sgRNA and DNA binding, the nuclease lobe contains the HNH and RuvC nuclease domains, respectively, appropriately positioned for cleavage of the complementary and non-complementary strands of the target DNA. The nuclease lobe also contains a carboxyl-terminal domain responsible for interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analysis reveal the molecular mechanism of RNA-guided DNA targeting by Cas9 and thus pave the way for the rational design of new, versatile genome editing technologies.

[0218] Wu et al. mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with a single guide RNA (sgRNA) in mouse embryonic stem cells (mESCs). The authors showed that each of four test sgRNAs targeted dCas9 to tens to thousands of genomic sites frequently characterized by a 5-nucleotide seed region and an NGG protospacer adjacent motif (PAM) in the sgRNA. Chromatin inaccessibility reduced dCas9 binding to other sites with matching seed sequences; therefore, 70% of off-target sites were associated with genes. The authors showed that targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically inactive Cas9 identified only one mutation site above background. The authors proposed a two-state model for Cas9 binding and cleavage in which a seed match triggers binding, but extensive pairing with the target DNA is required for cleavage.

[0219] Hsu 2014 is a review article that discusses the history of CRISPR-Cas9 from yogurt to genome editing, including genetic screening of cells generally, that is present in the information, data, and knowledge in applications filed prior to June 5, 2014, which are part of the series of applications herein. The general teachings of Hsu 2014 do not include the defined models, animals, herein.

[0220] Also included is Tsai et al., "Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing," Nature Biotechnology 32(6):569-77 (2014), which is not considered prior art to the present invention or application, but may be considered in the practice of the present invention.

[0221] Generally, CRISPR-Cas or CRISPR system, as used in the above-mentioned documents, e.g., International Publication No. WO 2014 / 093622 (PCT / US2013 / 074667), refers collectively to transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, e.g., sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portion tracrRNA), tracr mate sequences (including "direct repeats" and tracrRNA processing portion direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or "RNAs" as the term is used herein (e.g., RNAs for guiding Cas9, e.g., CRISPR RNA and trans-activating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)), or other sequences and transcripts from the CRISPR locus. Generally, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of target sequences (also referred to as protospacers in the context of endogenous CRISPR systems). In the context of CRISPR complex formation, "target sequence" refers to a sequence that guide sequences are designed to have such complementarity that hybridization between the target sequence and guide sequence promotes the formation of CRISPR complexes. Target sequences can include any polynucleotide, for example, DNA or RNA polynucleotides. In some embodiments, target sequences are located in the nucleus or cytoplasm of a cell. In some embodiments, direct repeats can be identified in silico by searching for repeat motifs that meet any or all of the following criteria: 1. found within a 2 Kb window of the genomic sequence flanking the type II CRISPR locus; 2. span from 20 to 50 bp; and 3. spaced by 20 to 50 bp. In some embodiments, two of these criteria can be used, for example, 1 and 2, 2 and 3, or 1 and 3.In some embodiments, all three criteria may be used.

[0222] 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 their analogs. Polynucleotides can have any three-dimensional structure and can 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 defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (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 can contain one or more modified nucleotides, such as methylated nucleotides or nucleotide analogs. Modifications to the nucleotide structure, if present, can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0223] In embodiments of the present invention, the terms "chimeric RNA," "chimeric guide RNA," "guide RNA," "single guide RNA," and "synthetic guide RNA" are used interchangeably to refer to a polynucleotide sequence comprising a guide sequence, a tracr sequence, and a tracr mate sequence. The term "guide sequence" refers to an approximately 20-bp sequence within the guide RNA that defines the target site and can be used interchangeably with the terms "guide" or "spacer." The term "tracr mate sequence" can also be used interchangeably with the term "direct repeat." An exemplary CRISPR-Cas system is illustrated in FIG. 1.

[0224] The term "wild-type" as used herein is a term of the art understood by those skilled in the art and means the typical form of an organism, strain, gene or characteristic as it occurs in nature, as distinguished from mutant or variant forms.

[0225] The term "variant" as used herein should be taken to mean a display of qualities having a pattern that deviates from that occurring in nature.

[0226] The terms "non-naturally occurring" and "engineered" are used interchangeably and refer to artificial involvement. When referring to a nucleic acid molecule or polypeptide, the term means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is naturally associated or found in the natural state.

[0227] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by classical Watson-Crick or other non-classical types. The percent complementarity indicates the percentage of residues in a nucleic acid molecule that 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 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" 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.

[0228] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence will predominantly hybridize to the target sequence and will not substantially hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on numerous factors. Generally, the longer the sequence, the higher the temperature at which the sequence will specifically hybridize to its target sequence. Non-limiting examples of stringent conditions are detailed in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology—Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay," Elsevier, NY.

[0229] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonding between the bases of nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex can contain two strands forming a double-stranded structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can 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 that can hybridize to a given sequence is referred to as the "complementary strand" of the given sequence.

[0230] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide can be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0231] 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, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have undergone modifications, such as 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 D- and L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0232] 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. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0233] The terms "therapeutic agent," "therapeutic drug," or "treatment agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. Beneficial effects include enabling a diagnostic measurement; ameliorating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or pathological condition; and generally neutralizing a disease, symptom, disorder, or pathological condition.

[0234] As used herein, "treatment" or "treating" or "alleviating" or "ameliorating" are used interchangeably. These terms refer to an approach to obtain a benefit or desired result, for example, but not limited to, a therapeutic benefit and / or a preventive benefit. A therapeutic benefit refers to any treatment-related improvement in or effect on one or more diseases, conditions, or symptoms being treated. For preventive benefit, the composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not previously manifested.

[0235] The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug sufficient to produce a benefit or desired result. The therapeutically effective amount may vary depending on one or more of the subject and condition being treated, the subject's weight and age, the severity of the condition, the mode of administration, etc., which can be easily determined by those skilled in the art. This term also applies to the dose that provides an image for detection by any one of the imaging methods described herein. The specified dose may vary depending on one or more of the specific drug selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.

[0236] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of one in the art. See Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M.A.usubel, et al. eds., (1987)); 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)).

[0237] Some embodiments of the present invention relate to vector systems containing one or more vectors, or to vectors themselves.Vector can be designed for the 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 further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Alternatively, recombinant expression vectors can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.

[0238] Vectors can be introduced into and propagated within prokaryotes. In some embodiments, prokaryotes are used to amplify copies of vectors to be introduced into eukaryotic cells or as intermediate vectors in the production of vectors to be introduced into eukaryotic cells (e.g., amplifying plasmids as part of a viral vector packaging system). In some embodiments, prokaryotes are used to amplify copies of vectors, express one or more nucleic acids, and provide a source of one or more proteins, for example, for delivery to a host cell or host organism. Protein expression in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add multiple amino acids to the encoded protein, for example, to the amino terminus of the recombinant protein. Such fusion vectors can serve one or more purposes, for example, (i) increasing the expression of the recombinant protein; (ii) increasing the solubility of the recombinant protein; and (iii) aiding in the purification of the recombinant protein by acting as a ligand in affinity purification. In fusion expression vectors, a proteolytic cleavage site is often introduced at the junction of the fusion moiety and the recombinant protein to allow separation of the recombinant protein from the fusion moiety after purification of the fusion protein. Such enzymes and their cognate recognition sequences include factor Xa, thrombin, and enterokinase. Exemplary 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, NJ), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A to the target recombinant protein, respectively.

[0239] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0240] In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6:229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30:933-943), pJRY88 (Schultz et al., 1987. Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[0241] In some embodiments, the vector drives protein expression in insect cells using a baculovirus expression vector. Baculovirus vectors available for protein expression 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).

[0242] In some embodiments, the vector may drive 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, expression vector control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus type 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, for example, 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, NY, 1989.

[0243] In some embodiments, the recombinant mammalian expression vector can direct 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), promoters of T-cell receptors in particular (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), pancreatic-specific promoters (Edlund, et al., 1989, Proc. Natl. Acad. Sci. USA 86:5473-5477), and the like. et al., 1985, Science 230:912-916), and mammary gland-specific promoters (e.g., whey promoters; U.S. Pat. No. 4,873,316 and European Patent Application Publication No. 264,166). Developmentally regulated promoters, such as murine hox promoters (Kessel and Gruss, 1990, Science 249:374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989, Genes Dev. 3:537-546), are also encompassed.

[0244] In some embodiments, the regulatory element is operably linked to one or more elements of the CRISPR system, so as to drive the expression of one or more elements of the CRISPR system.Generally, CRISPR (Clustered Regularly Interspaced Short Repeats), also known as SPIDR (Spacer Interspersed Direct Repeat), constitutes a family of DNA loci that are usually specific to certain bacterial species.CRISPR loci include a distinct class of interspersed short sequence repeats (SSRs) recognized in Escherichia coli (E. coli) (Ishino et al., J. Bacteriol.,169:5429-5433

[1987] ; and Nakata et al., J. Bacteriol.,171:3553-3556

[1989] ) and related 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] ). CRISPR loci typically differ from other SSRs in the structure of their repeats, which are termed short regularly interspaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33

[2002] ; and Mojica et al., Mol. Microbiol., 36:244-246

[2000] ). Generally, repeats are short elements that occur in clusters regularly spaced by unique intervening sequences of substantially constant length (Mojica et al.,

[2000] , supra). Repeat sequences are highly conserved between strains, butThe number of interspersed repeats and the sequence of the spacer region typically vary from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401

[2000] ). CRISPR loci have been identified in over 40 prokaryotes (e.g., Jansen et al., Mol. Microbiol., 43:1565-1575

[2002] ; and Mojica et al. See, e.g., et al.,

[2005] , including, but not limited to, the genera Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, and the like. fex), Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter,The genera Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

[0245] Generally, a "CRISPR system" collectively refers to transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, e.g., sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system are derived from a particular organism that contains an endogenous CRISPR system, e.g., Streptococcus pyogenes. Generally, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at target sequences (also referred to as protospacers in endogenous CRISPR systems). With regard to the formation of CRISPR complexes, "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided that there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. The target sequence can comprise any polynucleotide, for example, a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence can be present in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used for recombination into a targeted locus containing a target sequence is referred to as an "editing template," "editing polynucleotide," or "editing sequence." In an embodiment of the present invention, an exogenous template polynucleotide can be referred to as an editing template. In one aspect of the invention, the recombination is homologous recombination.

[0246] Typically, with respect to endogenous CRISPR systems, 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 the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs therefrom). Without being bound by theory, the tracr sequence may comprise or consist of all or a portion of the wild-type tracr sequence (e.g., more than about or about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence), and may also form part of a CRISPR complex, for example, by hybridization along at least a portion of the tracr sequence with all or a portion of a tracr mate sequence operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex. As with the target sequence, perfect complementarity is not required, provided that sufficient complementarity exists to be functional. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more vectors driving the expression of one or more elements of a CRISPR system are introduced into a host cell, such that the expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. For example, the Cas enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector, and one or more additional vectors providing any component of the CRISPR system are not included in the first vector.The CRISPR system elements combined in a single vector can be arranged in any suitable orientation; for example, an element can be located 5' (upstream) or 3' (downstream) relative to a second element. The coding sequence of an element can be located on the same or opposite strand as the coding sequence of a second element and can be oriented in the same or opposite direction. In some embodiments, a single promoter drives the expression of a transcript encoding a CRISPR enzyme and one or more of a guide sequence, a 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. A single vector construct for SpCas9 is shown in Figure 22.

[0247] In some embodiments, a vector comprises one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). 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 after insertion of the 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 located between two tracr mate sequences to allow insertion of a guide sequence at the respective 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 a combination thereof. When using multiple different guide sequences, can use a single expression construct to target the CRISPR activity to multiple different corresponding target sequences in cells.For example, a single vector can comprise about or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more guide sequences.In some embodiments, about or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of these guide sequence-containing vectors can be provided and optionally delivered into cells.

[0248] In some embodiments, the vector comprises a regulatory element operably linked to an enzyme coding sequence that encodes a CRISPR enzyme, e.g., 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, Csel, 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 Streptococcus pyogenes (S. pyogenes) Cas9 protein can be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, for example, Cas9. In some embodiments, the CRISPR enzyme is Cas9, and can be Cas9 from Streptococcus pyogenes (S. pyogenes) or Streptococcus pneumoniae (S. pneumoniae). In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of the target sequence, for example, within the target sequence and / or the complementary strand 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 the target sequence. In some embodiments, the vector encodes a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence.For example, an aspartate-to-alanine substitution (D10A) in the RuvCI catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaving a single strand). Other examples of mutations that convert Cas9 into a nickase include, but are not limited to, H840A, N854A, and N863A. In embodiments of the present invention, nickases can be used for genome editing via homologous recombination. For example, Figure 21 illustrates genome editing via homologous recombination. Figure 21(a) shows a schematic diagram of SpCas9 nickase with a D10A mutation in the RuvCI catalytic domain. (b) Schematic diagram depicting homologous recombination (HR) at the human EMX1 locus using a sense or antisense single-stranded oligonucleotide as a repair template. (c) Sequence of the region modified by HR. d, SURVEYOR assay (n=3) for wild-type (wt) and nickase (D10A) SpCas9-mediated indels at the EMX1 target 1 locus. Arrows indicate the positions of predicted fragment sizes.

[0249] In some embodiments, Cas9 nickase can be used in combination with a guide sequence, e.g., two guide sequences targeting the sense and antisense strands of a DNA target, respectively. This combination allows both strands to be nicked and used to induce NHEJ. Applicants have demonstrated the efficacy of two nickase targets (i.e., sgRNAs targeted to different strands of DNA at the same location) in inducing mutagenic NHEJ (data not shown). While a single nickase (Cas9-D10A with a single sgRNA) cannot induce NHEJ and create indels, applicants have shown that a dual 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 approximately 50% of that of a nuclease (i.e., regular Cas9 without the D10 mutation) in hESCs.

[0250] As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) can be mutated to produce a mutant Cas9 that substantially lacks all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme that substantially lacks all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity if the DNA cleavage activity of the mutant enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of its non-mutated form. Other mutations may be useful; if the Cas9 or other CRISPR enzyme is from a species other than S. pyogenes, corresponding amino acid mutations can be made to achieve similar effects.

[0251] In some embodiments, the enzyme coding sequence encoding CRISPR enzyme is codon-optimized for expression in specific cells, for example, eukaryotic cells.Eukaryotic cells can be derived from or derived from specific organisms, for example, mammals, for example, but not limited to, humans, mice, rats, rabbits, dogs, or non-human primates.Generally, codon optimization refers to the process of modifying nucleic acid sequences to improve expression in target host cells by replacing at least one codon (for example, about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of native sequence with the more frequently or most frequently used codon in the gene of the host cell, while maintaining the native amino acid sequence.Different species show specific bias for certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, among other things, on the characteristics of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of tRNAs selected in a cell generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in "codon usage databases," 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 specific sequences for expression in specific host cells are also available, for example, in GeneForge (Aptagen; Jacobus, PA).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.

[0252] Generally, a guide sequence is any polynucleotide sequence that has 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 is greater than about or about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any suitable algorithm for aligning sequences, including, but not limited to, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, CA), and others. Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is about or greater 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, the guide sequence is about 75, 50, 45, 40, 35, 30, 25 The length of the guide sequence is less than 1, 20, 15, 12 or less nucleotides.The ability of the guide sequence to direct the sequence-specific binding of CRISPR complex to target sequence can be evaluated by any suitable assay.For example, the components of the CRISPR system sufficient to form a CRISPR complex, for example, the guide sequence to be tested, can be provided to the host cell having the corresponding target sequence, for example, by transfection with the vector encoding the components of the CRISPR sequence, and then the preferential cleavage within the target sequence is evaluated, for example, by the Surveyor assay described herein.Similarly, cleavage of a target polynucleotide sequence can be assessed in a test tube by providing the target sequence, a component of a CRISPR complex, for example, a guide sequence to be tested and a control guide sequence that differs from the test guide sequence, and comparing the rate of binding or cleavage at the target sequence between the test and control guide sequence reactions. Other assays are contemplated and will be recognized by those skilled in the art.

[0253] The guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for S. pyogenes Cas9, a unique target sequence in the genome can include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG, where NNNNNNNNNNNNXGG (N is A, G, T, or C; X can be anything) has a single occurrence in the genome. A unique target sequence in the genome can include a S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG, where NNNNNNNNNNNXGG (N is A, G, T, or C; X can be anything) has a single occurrence in the genome. For S. thermophilus CRISPR1Cas9, a unique target sequence in the genome can include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; W is A or T) has a single occurrence in the genome. A unique target sequence in the genome can include a S. thermophilus CRISPR1Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; W is A or T) has a single occurrence in the genome. For S. pyogenes Cas9, a unique target sequence in the genome can include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; X can be anything) has a single occurrence in the genome.A unique target sequence in a genome can include a S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG, where NNNNNNNNNNNXGGXG (N is A, G, T, or C; X can be anything) has a single occurrence in the genome. In each of these sequences, "M" can be A, G, T, or C and need not be considered in identifying the sequence as unique.

[0254] In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. An example of such an algorithm is mFold, described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is the online web server RNAfold, developed by the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (see, for example, A.R. Gruber et al., 2008, Cell 106(1):23-24; and P.A. Carr and G.M. Church, 2009, Nature Biotechnology 27(12):1151-62). Further algorithms can be found in U.S. Patent Application No. 61 / 836,080 (Attorney Docket No. 44790.11.2022; Broad Reference No. BI-2013 / 004A), which is incorporated herein by reference.

[0255] Generally, the tracr mate sequence includes any sequence that has sufficient complementarity with the tracr sequence to promote one or more of the following: (1) excision of the guide sequence flanked by the tracr mate sequence in cells containing the corresponding tracr sequence; and (2) formation of a CRISPR complex in the target sequence (the CRISPR complex includes the tracr mate sequence hybridized to the tracr sequence). Generally, the degree of complementarity is based on optimal alignment of the tracr mate sequence and the tracr sequence along the shorter length of the two sequences. Optimal alignment can be determined by any suitable alignment algorithm and can further account for secondary structures, such as self-complementarity within the tracr sequence or the tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and the tracr mate sequence along the shorter length of the two sequences is greater than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or more when optimally aligned. An exemplary illustration of optimal alignment between the tracr sequence and the tracr mate sequence is provided in Figures 10B and 11B. In some embodiments, the tracr sequence is about or greater 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 the tract mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript with a secondary structure, e.g., a hairpin. A preferred loop-forming sequence used in the hairpin structure is 4 nucleotides in length and most preferably has the sequence GAAA. However, longer or shorter loop sequences can be used, as can alternative sequences. The sequence preferably includes a nucleotide triplet (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In one embodiment of the present 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 another further embodiment of the present invention, the transcript has at most five hairpins. In some embodiments, the single transcript further comprises a transcription termination sequence; preferably, this is a poly-T sequence, e.g., six T nucleotides. An illustrative illustration of such a hairpin structure is provided in the lower portion of Figure 11B, where the final "N" and the portion of the sequence 5' upstream of the loop correspond to the tracr mate sequence, and the portion of the sequence 3' upstream 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 the base of the guide sequence, the first block of lowercase letters represents the tracr mate sequence, the second block of lowercase letters represents the tracr sequence, and the final poly-T sequence represents the transcription terminator: [ka] In some embodiments, sequences (1) through (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) through (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from the transcript containing the tracr mate sequence (e.g., as illustrated in the top portion of Figure 11B).

[0256] In some embodiments, the CRISPR enzyme is part of a fusion protein containing 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 other domains of the CRISPR enzyme). The CRISPR enzyme fusion protein can include any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains with 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) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. 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, such as blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, such as, but not limited to, maltose binding protein (MBP), S-tags, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of fusion proteins comprising CRISPR enzymes are described in U.S. Patent Application Publication No. 20110059502, which is incorporated herein by reference. In some embodiments, tagged CRISPR enzymes are used to identify the location of the target sequence.

[0257] In one embodiment of the present invention, reporter genes, including but 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, such as blue fluorescent protein (BFP), can be introduced into cells to encode gene products that serve as markers for measuring altered or modified expression of the gene product. In a further embodiment of the present invention, a DNA molecule encoding the gene product can be introduced into cells via a vector. In a preferred embodiment of the present invention, the gene product is luciferase. In a further embodiment of the present invention, expression of the gene product is reduced.

[0258] In some embodiments, the present invention provides methods comprising delivering one or more polynucleotides, such as, for example, one or more vectors described herein, one or more transcripts thereof, and / or one or more proteins transcribed therefrom, to a host cell. In some embodiments, the present invention further provides cells produced by such cells, and organisms (e.g., 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 the cell. Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into 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 into a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses with episomal or integrated genomes after delivery to the cell.For an overview 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 See Immunology, Doerfler and Boehm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0259] Non-viral nucleic acid delivery methods include lipofection, nucleofection, microinjection, gene gun, virosome, liposome, immunoliposome, polycation or lipid:nucleic acid conjugate, naked DNA, artificial virion, and drug-enhanced DNA uptake.Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are commercially available (for example, Transfectam™ and Lipofectin™).Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91 / 17424; WO 91 / 16024.Delivery can be to cells (for example, in vitro or ex vivo administration) or target tissue (for example, in vivo administration).

[0260] The preparation of lipid:nucleic acid complexes, including targeted liposomes, e.g., immunolipid complexes, is well known to those skilled 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); see U.S. Patent 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).

[0261] The use of RNA or DNA virus-based systems for nucleic acid delivery takes advantage of a highly evolved process that targets viruses to specific cells in the body and transports the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or used to treat cells in vitro, and the modified cells can then be administered to patients (ex vivo). Conventional virus-based systems include retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors for gene transfer. Integration into the host genome is possible with retroviral, lentiviral, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiencies have been observed in many different cell types and target tissues.

[0262] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. Therefore, the choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors contain cis-acting long terminal repeats (LTRs) that have packaging capacity for foreign sequences up to 6-10 kb. The minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate therapeutic genes into target cells to provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), or 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, adenovirus-based systems can be used. Adenovirus-based vectors can exhibit extremely high transduction efficiency in many cell types and do not require cell division. High titers and expression levels have been obtained with such vectors. The vectors can be produced in large quantities using a relatively simple system.For example, adeno-associated virus ("AAV") vectors can also be used to transduce cells with target nucleic acids 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). The construction of recombinant AAV vectors is described in numerous publications, e.g., U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al. 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).

[0263] Typically, packaging cells are used to form viral particles that can infect host cells. Such cells include 293 cells, which package adenovirus, and ψ2 or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually produced by creating cell lines that package nucleic acid vectors into viral particles. The vector typically contains the minimum viral sequences required for packaging and subsequent integration into the host, with other viral sequences replaced by an expression cassette for the polynucleotide to be expressed. Defective viral functions are typically provided in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the ITR sequences from the AAV genome required for packaging and integration into the host genome. Viral DNA is packaged into a cell line containing a helper plasmid encoding other AAV genes, i.e., rep and cap, but lacking the ITR sequences. The cell line can also be infected with adenovirus as a helper. Helper virus promotes the replication of AAV vector and the expression of AAV gene from helper plasmid.Helper plasmid is not packaged in significant amounts due to the lack of ITR sequence.Contamination by adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.Additional methods for delivering nucleic acid to cells are known to those skilled in the art.For example, see US Patent Application Publication No. 20030087817, which is incorporated herein by reference.

[0264] In some embodiments, host cells are transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, cells are transfected as they naturally occur in a subject. In some embodiments, the transfected cells are harvested from a subject. In some embodiments, the cells are derived from cells harvested from a subject, e.g., cell lines. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, and Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, BALB / 3T3 mouse embryonic fibroblasts, 3T3Swiss, 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, BR29 3, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku 812, KCL22, KG1, KYO1, LNCap, Ma-Mel1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR / 0.2R, MONO-MAC6, 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 / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic variants thereof. Cell lines are available from a variety of sources known to those of skill in the art (see, e.g., American Type Culture Collection (ATCC), Manassus, Va.). In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines comprising one or more vector-derived sequences. In some embodiments, cells transiently transfected with components of a CRISPR system described herein (e.g., by transient transfection of one or more vectors or transfection with RNA) and modified through the activity of a CRISPR complex are used to establish new cell lines comprising cells containing the modification but lacking any other exogenous sequences. 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 the evaluation of one or more test compounds.

[0265] In some embodiments, one or more vectors described herein are used to produce non-human transgenic animals or transgenic plants. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, or rabbit. In some embodiments, the organism or subject is a plant. In some embodiments, the organism, subject, or plant is algae. Methods for producing transgenic plants and animals are known in the art and generally begin, for example, with the cell transfection methods described herein. Transgenic animals are also provided, as are transgenic plants, particularly crops and algae. Transgenic animals or plants may be useful for purposes other than providing disease models. These may include, for example, food or feed production through the expression of higher protein, carbohydrate, nutrient, or vitamin levels than those normally found in wild-type plants. In this regard, transgenic animals, particularly legumes and tubers, and animals, particularly mammals, such as livestock (cattle, sheep, goats, and pigs), are preferred, although poultry and edible insects are also preferred.

[0266] Transgenic algae or other plants, such as rapeseed, can be particularly useful, for example, in the production of vegetable oils or biofuels, such as alcohols (especially methanol and ethanol). They can be engineered to express or overexpress high levels of oils or alcohols used in the oil or biofuel industries.

[0267] In one aspect, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell, which can be in vivo, ex vivo, or in vitro. In some embodiments, the method comprises sampling a cell or cell population from a human or non-human animal or plant (including microalgae) and modifying one or more cells. Culturing can be carried out ex vivo at any stage. One or more cells can even be reintroduced into a non-human animal or plant (including microalgae).

[0268] In one aspect, the present invention provides a method for modifying target polynucleotide in eukaryotic cells.In some embodiments, the method comprises: allowing CRISPR complex to bind to target polynucleotide, causing the cleavage of said target polynucleotide, thereby modifying said target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with the guide sequence that hybridizes with the target sequence in said target polynucleotide, and said guide sequence is then bound to the tracr mate sequence that hybridizes with the tracr sequence.

[0269] In one aspect, the present invention provides a method for modifying the expression of polynucleotide in eukaryotic cells.In some embodiments, the method comprises: allowing CRISPR complex to bind to polynucleotide, and thereby causing the binding to increase or decrease the expression of said polynucleotide; CRISPR complex comprises CRISPR enzyme complexed with guide sequence that hybridizes with target sequence in said target polynucleotide, and said guide sequence is then bound to tracr mate sequence that hybridizes with tracr sequence.

[0270] With recent advances in crop genomics, the ability to use CRISPR-Cas systems to perform efficient and cost-effective gene editing and manipulation allows for the rapid selection and comparison of single and multiplexed genetic manipulations to transform such genomes for improved production and trait enhancement. In this regard, reference is made to U.S. patents and publications: U.S. Patent No. 6,603,061 - Agrobacterium-Mediated Plant Transformation Method; U.S. Patent No. 7,868,149 - Plant Genome Sequences and Uses Thereof and U.S. Patent Application Publication No. 2009 / 0100536 - Transgenic Plants with Enhanced Agronomic Traits, the entire contents and disclosures of each of which are incorporated herein by reference in their entirety. In implementing the present invention, the contents and disclosures of Morrell et al. "Crop genomics: advances and applications" Nat Rev Genet. 2011 Dec 29; 13(2): 85-96 are also incorporated herein by reference in their entirety. In an advantageous embodiment of the present invention, CRISPR / Cas9 system is used to engineer microalgae.The CRISPR-Cas system can be used in plant systems as well, as provided in the manuscript "Efficient Genome Editing in Plants using a CRISPR / Cas System" by Feng et al.Cell Res.2013 Aug 20.doi:10.1038 / cr.2013.114.[Epub ahead of print], which is incorporated by reference in its entirety, demonstrating that engineered CRISPR / Cas complexes can be used to create double-strand breaks at specific sites in plant genomes to achieve targeted genome modification in dicotyledonous and monocotyledonous plants.Therefore, unless otherwise clear, any reference to animal cells herein can also apply mutatis mutandis to plant cells.

[0271] In plants, pathogens are often host-specific. For example, Fusarium oxysporum f.sp. lycopersici, which causes tomato wilt, attacks only tomatoes, and F. oxysporum f. dianthii and Puccinia graminis f.sp. tritici attack only wheat. Plants have pre-existing and induced defenses to resist many pathogens. Mutation and recombination events between plant generations, particularly as pathogens replicate at higher frequencies than plants, result in genetic variability that leads to susceptibility. Non-host resistance can also exist in plants, for example, when the host and pathogen are incompatible. Horizontal resistance, e.g., partial resistance to all pathogen strains, typically controlled by many genes, and vertical resistance, e.g., complete resistance to some pathogen strains but not others, typically controlled by a few genes, may also exist. At the gene-for-gene level, plants and pathogens coevolve, with genetic changes in one balancing out changes in the other. Thus, breeders use natural variability to combine the most useful genes for yield, quality, uniformity, cold tolerance, and resistance. Sources of resistance genes include native or exotic varieties, landraces, wild plant relatives, and induced mutations (e.g., treatment of plant material with mutagens). The present invention provides plant breeders with a novel means of inducing mutations. Thus, by analyzing the genomes of sources of resistance genes and using the present invention in varieties with desired characteristics or traits, those skilled in the art can induce the production of resistance genes with greater precision than previous mutagens, thereby accelerating and improving plant breeding programs.

[0272] In one aspect, the present invention provides a kit containing any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit includes a vector system and instructions for use of the kit. In some embodiments, the vector system includes: (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 (the guide sequence, when expressed, directs the sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, the CRISPR complex comprising (1) a guide sequence hybridized to the target sequence, and (2) a CRISPR enzyme complexed with the tracr mate sequence hybridized to the tracr sequence); and / or (b) a second regulatory element operably linked to an enzyme coding sequence encoding the CRISPR enzyme comprising a nuclear localization sequence. The elements can be provided individually or in combination, and can be provided in any suitable container, for example, a vial, a bottle, or a tube. In some embodiments, the kit includes instructions in one or more languages, for example, in two or more languages.

[0273] In some embodiments, the kit includes one or more reagents used in a process utilizing one or more of the elements described herein. The reagents can be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. The reagents can be provided in a form usable in a particular assay or in a form that requires the addition of one or more other components prior to use (e.g., a concentrate or lyophilized form). The buffer can be any buffer, including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10. In some embodiments, the kit includes one or more oligonucleotides corresponding to the guide sequence for insertion into a vector to operably link the guide sequence and regulatory elements. In some embodiments, the kit includes a homologous recombination template polynucleotide.

[0274] In one embodiment, the present invention provides a method for using one or more elements of the CRISPR system.The CRISPR complex of the present invention provides an effective means for modifying target polynucleotide.The CRISPR complex of the present invention has a wide range of uses, for example, modifying (for example, deletion, insertion, translocation, inactivation, activation) target polynucleotide in a large number of cell types.Therefore, the CRISPR complex of the present invention has a wide range of applications, for example, in gene therapy, drug screening, disease diagnosis and prognosis.An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence in a target polynucleotide.The guide sequence is then linked to a tracr mate sequence that hybridizes to a tract sequence.

[0275] The target polynucleotide of a CRISPR complex can be any polynucleotide that is endogenous or exogenous to eukaryotic cells. For example, the target polynucleotide can be a polynucleotide that remains in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence that encodes a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). The target can be a control element, a regulatory element, a promoter, an enhancer, or a silencer. In some embodiments, the promoter can be located in the region +200bp or even +1000bp from the TTS. In some embodiments, the regulatory region can be an enhancer. The enhancer can typically be located more than +1000bp from the TTS. More specifically, the expression of eukaryotic protein-coding genes is generally regulated through multiple cis-acting transcriptional control regions. Some control elements are located close to the start site (promoter-proximal elements), while other elements are located more distally (enhancers and silencers). Promoters determine the site of transcription initiation and direct the binding of RNA polymerase II. Three types of promoter sequences have been identified in eukaryotic DNA. TATA boxes are the most common and are frequently found in rapidly transcribed genes. Initiator promoters are found less frequently in some genes, and CpG islands are characteristic of transcribed genes. Promoter-proximal elements occur within approximately 200 base pairs of the initiation site. Some such elements contain up to approximately 20 base pairs and may serve to regulate specific genes. Enhancers are usually approximately 100-200 base pairs in length and contain multiple 8- to 20-bp regulatory elements. They can be located 200 base pairs to tens of kilobases upstream or downstream from the promoter, within introns, or downstream from the final exon of a gene. Promoter-proximal elements and enhancers can be cell-type specific, functioning only in specific differentiated cell types. However, any of these regions can also be target sequences and are encompassed by the concept that a target can be a control element or regulatory element or promoter or enhancer or silencer.

[0276] Without being bound by theory, it is believed that target sequence should be associated with PAM (protospacer adjacent motif); that is, the short sequence that is recognized by CRISPR complex.The exact sequence and length requirements for PAM vary depending on the CRISPR enzyme used, but PAM is typically a 2-5 base pair sequence adjacent to protospacer (that is, target sequence).Examples of PAM sequence are listed in the following example section, and those skilled in the art can identify other PAM sequences that are used for a given CRISPR enzyme.

[0277] Target polynucleotides for CRISPR complexes can include many of the disease-associated genes and polynucleotides and signaling biochemical pathway-associated genes and polynucleotides listed in U.S. Provisional Patent Applications Nos. 61 / 736,527 and 61 / 748,427 (both entitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed December 12, 2012 and January 2, 2013, respectively), having Broad Reference Nos. BI-2011 / 008 / WSGR Docket No. 44063-701.101 and BI-2011 / 008 / WSGR Docket No. 44063-701.102, respectively, the entire contents of which are incorporated herein by reference in their entireties.

[0278] Examples of target polynucleotides include sequences related to signaling biochemical pathways, such as signaling biochemical pathway-related genes or polynucleotides. Examples of target polynucleotides include disease-related genes or polynucleotides. "Disease-related" genes or polynucleotides refer to any gene or polynucleotide that produces transcription or translation products at abnormal levels or in abnormal forms in cells derived from diseased tissues compared with tissues or cells of non-disease control. This can be a gene that is expressed at abnormally high levels; or a gene that is expressed at abnormally low levels, and expression changes correlate with the occurrence and / or progression of disease. Disease-related genes also refer to genes that are directly responsible for the pathogenesis of disease or have mutations or genetic variations that are in linkage disequilibrium with genes that are responsible for the pathogenesis of disease. The transcription or translation products can be known or unknown, and can be at normal or abnormal levels.

[0279] Examples of disease-associated genes and polynucleotides are available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), and are available on the World Wide Web.

[0280] Examples of disease-associated genes and polynucleotides are listed in Tables A and B. Disease-specific information is available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), and is available on the World Wide Web. Examples of signaling biochemical pathway-associated genes and polynucleotides are listed in Table C.

[0281] The mutation in these genes and pathways can cause inappropriate protein production or inappropriate amount of protein, which affects function.More examples of genes, diseases and proteins are incorporated herein by reference from the United States provisional patent application.Such genes, proteins and pathways can be the target polynucleotide of CRISPR complex.

[0282] [Table 6]

[0283] [Table 7]

[0284] [Table 8]

[0285] [Table 9]

[0286] [Table 10]

[0287] Table 11

[0288] Table 12

[0289] Table 13

[0290] Table 14

[0291] Table 15

[0292] Table 16

[0293] Table 17

[0294] Table 18

[0295] Table 19

[0296] Table 20

[0297] Table 21

[0298] [Table 22]

[0299] Embodiments of the present invention also relate to methods and compositions related to gene knockout, gene amplification, and repair of specific mutations associated with DNA repeat instability and neurological diseases (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities and Neurological Diseases, Second Edition, Academic Press, October 13, 2011 - Medical). Tandem repeat sequences of defined nature have been found to be responsible for more than 20 human diseases (New insights into repeat instability: role of RNA-DNA hybrids. McIvor EI, Polak U, Napierala M. RNA Biol. 2010 September-Oct;7(5):551-8). The CRISPR-Cas system can be used to correct these abnormalities of genomic instability.

[0300] A further aspect of the present invention relates to the use of CRISPR-Cas systems to correct abnormalities in the EMP2A and EMP2B genes, which have been identified as being associated with Lafora disease. Lafora disease is an autosomal recessive condition characterized by progressive myoclonic epilepsy that may begin as epileptic seizures in adolescence. Some cases of the disease may be caused by mutations in as-yet-unidentified genes. The disease causes seizures, muscle spasms, difficulty walking, dementia, and ultimately death. Currently, no treatments have been proven effective against disease progression. Other genetic abnormalities associated with epilepsy can also be targeted using CRISPR-Cas systems, and the underlying genetics are further described in Genetics of Epilepsy and Genetic Epilepsies, edited by Giuliano Avanzini and Jeffrey L. Noebels, Mariani Foundation Paediatric Neurology: 20; 2009).

[0301] In yet another embodiment of the present invention, the CRISPR-Cas system can be used to correct eye defects resulting from several genetic mutations, as further described in Genetic Diseases of the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford University Press, 2012.

[0302] Some further aspects of the present invention relate to the correction of abnormalities associated with a wide range of genetic diseases, which are further described on the National Institutes of Health website (website at health.nih.gov / topic / GeneticDisorders) under the topic subsection Genetic Disorders. Genetic brain diseases 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 disease, Gerstmann-Straussler-Scheinker disease, Huntington's disease and other triplet repeat diseases, Leigh disease, Lesch-Nyhan syndrome, Menkes disease, mitochondrial myopathy, and NINDS colpocephaly. These diseases are further described on the National Institutes of Health website under the subsection Genetic Brain Disorders.

[0303] In some embodiments, the condition is neoplasia. In some embodiments, where the condition may be neoplasia, the gene to be targeted may be any of those listed in Table A (such as PTEN in this case). In some embodiments, the condition may be age-related macular degeneration. In some embodiments, the condition may be schizophrenia. 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-associated disorder. In some embodiments, the condition may be a prion-associated disorder. In some embodiments, the condition may be ALS. In some embodiments, the condition may be 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.

[0304] 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.

[0305] An example of a preference-related protein is ABAT.

[0306] Examples of inflammation-related proteins include monocyte chemoattractant protein-1 (MCP1) encoded by the Ccr2 gene, CC chemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, IgG receptor IIB (FCGR2b, also known as CD32) encoded by the Fcgr2b gene, or Fc epsilon R1g (FCER1g) protein encoded by the Fcer1g gene.

[0307] Examples of cardiovascular disease-related proteins include, for example, IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin I2 (prostacyclin) synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-binding cassette, subfamily G (WHITE), member 8), or CTSK (cathepsin K).

[0308] Examples of Alzheimer's disease-related proteins include, for example, the very low density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, the ubiquitin-like modifier activating enzyme 1 (UBA1) encoded by the UBA1 gene, or the NEDD8 activating enzyme E1 catalytic subunit protein (UBE1C) encoded by the UBA3 gene.

[0309] Examples of proteins associated with autism spectrum disorders include, for example, benzodiazepine receptor (peripheral)-associated protein 1 (BZRAP1) encoded by the BZRAP1 gene, AF4 / FMR2 family member 2 protein (AFF2) encoded by the AFF2 gene (also known as MFR2), fragile X mental retardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene, or fragile X mental retardation autosomal homolog 2 protein (FXR2) encoded by the FXR2 gene.

[0310] Examples of proteins associated with macular degeneration include the ATP-binding cassette subfamily A (ABC1) member 4 protein (ABCA4) encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded by the APOE gene, or the chemokine (CC motif) ligand 2 protein (CCL2) encoded by the CCL2 gene.

[0311] Examples of proteins associated with schizophrenia include NRG1, ErbB4, CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, and combinations thereof.

[0312] Examples of proteins involved in tumor suppression 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), Notch1, Notch2, Notch3, or Notch4.

[0313] Examples of proteins associated with secretase disorders can include, for example, PSENEN (presenilin enhancer 2 homolog (C. elegans)), CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid beta (A4) precursor protein), APH1B (anterior pharyngeal defect 1 homolog B (C. elegans)), PSEN2 (presenilin 2 (Alzheimer's disease 4)), or BACE1 (beta-site APP cleaving enzyme 1).

[0314] Examples of proteins associated with amyotrophic lateral sclerosis include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof.

[0315] Examples of proteins associated with prion diseases can include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof.

[0316] Examples of proteins associated with neurodegenerative pathology in prion disorders include, for example, A2M (alpha-2-macroglobulin), AATF (apoptosis antagonistic transcription factor), ACPP (prostatic acid phosphatase), ACTA2 (aortic smooth muscle actin alpha 2), ADAM22 (ADAM metallopeptidase domain), ADORA3 (adenosine A3 receptor), or ADRA1D (alpha-1D adrenergic receptor for alpha-1D adrenoceptor).

[0317] Examples of proteins associated with immunodeficiency include, for example, A2M [alpha-2-macroglobulin]; AANAT [arylalkylamine N-acetyltransferase]; ABCA1 [ATP-binding cassette subfamily A (ABC1), member 1]; ABCA2 [ATP-binding cassette subfamily A (ABC1), member 2]; or ABCA3 [ATP-binding cassette subfamily A (ABC1), member 3].

[0318] Examples of proteins associated with trinucleotide repeat disorders include, for example, AR (androgen receptor), FMR1 (Fragile X Mental Retardation 1), HTT (Huntington's), or DMPK (Myotonic Dystrophy Protein Kinase), FXN (Frataxin), and ATXN2 (Ataxin 2).

[0319] Examples of proteins associated with impaired neurotransmission include, for example, SST (somatostatin), NOS1 (nitric oxide synthase 1 (neuronal type)), ADRA2A (adrenergic alpha-2A receptor), ADRA2C (adrenergic alpha-2C receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-hydroxytryptamine (serotonin) receptor 2C).

[0320] Examples of neurodevelopment-related sequences include, for example, A2BP1 [ataxin 2-binding protein 1], AADAT [aminoadipate aminotransferase], AANAT [arylalkylamine N-acetyltransferase], ABAT [4-aminobutyrate aminotransferase], ABCA1 [ATP-binding cassette subfamily A (ABC1) member 1], or ABCA13 [ATP-binding cassette subfamily A (ABC1) member 13].

[0321] Further examples of preferred conditions treatable by the system of the present invention can be selected from the following: Alcardi-Goutières syndrome; Alexander disease; Allan-Herndon-Dudley syndrome; POLG-related disorders; alpha-mannosidosis (types II and III); Alström syndrome; Angelman syndrome; ataxia-telangiectasia; neuronal ceroid lipofuscinosis; beta-thellasamia; bilateral optic atrophy and (infantile) optic atrophy type 1; retinoblastoma (bilateral); Canavan disease; cerebro-ocular-facial-skeletal syndrome 1 [COFS1]; cerebrotendinous xanthomas Cornelia de Lange syndrome; MAPT-related disorders; inherited prion diseases; Dravet syndrome; early-onset familial Alzheimer's disease; Friedreich ataxia [FRDA]; Flins syndrome; fucosidosis; Fukuyama-type congenital muscular dystrophy; galactosialidosis; Gaucher disease; organic acidemia; hemophagocytic lymphohistiocytosis; Hutchinson-Gilford progeria syndrome; mucolipidosis type II; infantile free sialic acid storage disease; PLA2G6-associated neurodegeneration; Jervell-Lange-Nielsen syndrome; junctional epidermolysis bullosa; Huntington's disease; Krabbe disease (infantile form) ; Mitochondrial DNA-related Leigh syndrome and NARP; Lesch-Nyhan syndrome; LIS1-related lissencephaly; Lowe syndrome; Maple syrup urine disease; MECP2 duplication syndrome; ATP7A-related copper transport disorders; LAMA2-related muscular dystrophy; Arylsulfatase A deficiency; Mucopolysaccharidosis type I, II, or III; Peroxisomal biogenesis disorders, Zellweger syndrome spectrum; Neurodegeneration with cerebral iron storage; Acid sphingomyelinase deficiency; Niemann-Pick disease type C; Glycine encephalopathy; ARX-related disorders; Urea cycle disorders; COL1A1 / 2-related diaphyseal dysplasia; mitochondrial DNA deletion syndrome; PLP1-related disorders; Perry syndrome; Phelan-McDermott syndrome; glycogen storage disease type II (Pompe disease) (infantile form); 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; childhood-onset spinocerebellar ataxia; hexosaminidase A deficiency; lethal dysplasia type 1; collagen type VI-related disorders; Usher syndrome type 1; congenital muscular dystrophy;Wolf-Hirschhorn syndrome; lysosomal acid lipase deficiency; and xeroderma pigmentosum.

[0322] It is clear that it is envisioned that the system of the present invention can be used to target any target polynucleotide sequence.Some examples of pathological conditions or diseases that can be usefully treated using the system of the present invention are included in the table above, and examples of genes currently associated with these pathological conditions are also provided in the table.However, the genes exemplified are not exclusive.

[0323] The present invention also encompasses the delivery of engineered and optimized CRISPR-Cas system.Can use adeno-associated virus (AAV), lentivirus, adenovirus or other plasmid or virus vector types to deliver Cas9 and one or more guide RNAs, specifically using the formulation and dosage of, for example, US Patent No. 8,454,972 (the formulation, dosage for adenovirus), US Patent No. 8,404,658 (the formulation, dosage for AAV) and US Patent No. 5,846,946 (the formulation, dosage for DNA plasmid), and the formulation and dosage of the clinical trials involving lentivirus, AAV and adenovirus and the literature related to such clinical trials.For example, for AAV, the administration route, formulation and dosage can be as in US Patent No. 8,454,972 and as in the clinical trials involving AAV. For adenovirus, the route of administration, formulation, and dosage can be as described in U.S. Patent No. 8,404,658 and as described in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation, and dosage can be as described in U.S. Patent No. 5,846,946 and as described in clinical trials involving plasmids. Dosages may be based on or extrapolated to an average 70 kg individual and can be adjusted for patients, subjects, or mammals of different weights and species. Dosage frequency is within the discretion of a medical or veterinary practitioner (e.g., physician, veterinarian), depending on the usual factors, including the patient's or subject's age, sex, general health, other conditions, and the specific condition or symptom being addressed.

[0324] The viral vector can be injected into the tissue of interest.For cell type-specific genome modification, Cas9 expression can be driven by cell type-specific promoter.For example, liver-specific expression can use albumin promoter, and neuron-specific expression can use synapsin I promoter.

[0325] Transgenic Animals and Plants Transgenic animals are also provided. Preferred examples include animals containing Cas9 in the sense of the Cas9-encoding polynucleotide or protein itself. Mice, rats, and rabbits are preferred. To generate transgenic mice with the constructs exemplified herein, pure linear DNA can be injected into the pronuclei of zygotes from pseudopregnant females, e.g., CB56 females. Founders can then be identified, genotyped, and backcrossed with CB57 mice. The constructs can then be cloned and optionally verified, e.g., by Sanger sequencing. Knockouts are contemplated, e.g., when one or more genes are knocked out in the model. However, knockins (alone or in combination) are also contemplated. Exemplary knockin Cas9 mice have been generated and are exemplified, but Cas9 knockins are preferred. To generate Cas9 knockin mice, the same constitutive and conditional constructs can be targeted to the Rosa26 locus as described herein (Figures 25A-B and 26). The methods of U.S. Patent Application Publication Nos. 20120017290 and 20110265198, assigned to Sangamo BioSciences, Inc., for targeting the Rosa locus may be modified to utilize the CRISPR Cas system of the present invention. In another embodiment, the methods of U.S. Patent Application Publication No. 20130236946, assigned to Cellectis, for targeting the Rosa locus may also be modified to utilize the CRISPR Cas system of the present invention.

[0326] Usefulness of conditional Cas9 mice: We demonstrate that a Cas9 conditional expression construct can be activated by coexpression with Cre in 293 cells. We also demonstrate that properly targeted R1 mESCs can have active Cas9 when Cre is expressed. Because Cas9 is followed by a P2A peptide cleavage sequence and then EGFP, we identify successful expression by observing EGFP. We demonstrate Cas9 activation in mESCs. This same concept makes conditional Cas9 mice extremely useful. We can cross these conditional Cas9 mice with mice that ubiquitously express Cre (ACTB-Cre strain) to obtain mice that express Cas9 in all cells. Delivery of chimeric RNA should be all that is required to induce genome editing in fetal or adult mice. Interestingly, when conditional Cas9 mice are bred with mice expressing Cre under the control of a tissue-specific promoter, Cas9 should be present only in tissues that also express Cre. This approach could be used to restrict genome editing to precise tissues by delivering chimeric RNA to those tissues.

[0327] As mentioned above, transgenic animals are also provided, as well as transgenic plants, particularly crops and algae.Transgenic plants can be useful in applications other than providing disease models.Such applications can include, for example, food or feed production by expressing protein, carbohydrate, nutrient or vitamin levels higher than those normally found in wild-type plants.In this regard, transgenic plants, particularly legumes and tubers, and animals, particularly mammals such as livestock (dairy cows, sheep, goats and pigs), as well as poultry and edible insects, are also preferred.

[0328] Transgenic algae or other plants, such as rapeseed, can be particularly useful, for example, in the production of vegetable oils or biofuels, such as alcohols (especially methanol and ethanol). They can be engineered to express or overexpress high levels of oils or alcohols used in the oil or biofuel industry.

[0329] Adeno-associated virus (AAV) In terms of in vivo delivery, AAV is advantageous compared to other viral vectors for several reasons: Low toxicity (this may be due to the purification method not requiring ultracentrifugation of cellular particles that may activate the immune response) It is unlikely to cause insertional mutagenesis because it does not integrate into the host genome.

[0330] AAV has a packaging limit of 4.5 or 4.75 Kb. This means that Cas9, a promoter, and a transcription terminator must all be packaged in the same viral vector. Constructs larger than 4.5 or 4.75 Kb can cause a significant decrease in virus production. SpCas9 is very large, with the gene itself exceeding 4.1 Kb, making it difficult to package into AAV. Therefore, embodiments of the present invention include utilizing shorter Cas9 homologs. For example:

[0331] [Table 23]

[0332] Therefore, these species are generally preferred Cas9 species. Applicants present delivery and in vivo mouse brain Cas9 expression data.

[0333] Two methods of packaging Cas9-encoding nucleic acid molecules, e.g., DNA, into viral vectors to mediate genome modification in vivo are preferred: To achieve NHEJ-mediated gene knockout: Single viral vector: Vectors containing two or more expression cassettes: Promoter-Cas9-encoding nucleic acid molecule-terminator Promoter-gRNA1-terminator Promoter-gRNA2-terminator Promoter-gRNA(N)-terminator (up to the size limit of the vector) Dual viral vectors: Vector 1 contains one expression cassette to drive Cas9 expression. Promoter-Cas9-encoding nucleic acid molecule-terminator Vector 2 contains another expression cassette to drive expression of one or more guide RNAs. Promoter-gRNA1-terminator Promoter-gRNA(N)-terminator (up to the size limit of the vector)

[0334] To mediate homology-directed repair. In addition to the single and dual viral vector approaches described above, additional vectors are used to deliver the homology-directed repair template.

[0335] Promoters used to drive expression of Cas9-encoding nucleic acid molecules can include: AAV ITRs can act as promoters; this is advantageous as it eliminates the need for additional promoter elements, which can take up space in the vector. The additional space freed up can be used to drive expression of additional elements (such as gRNAs). Furthermore, because ITR activity is relatively weak, it can also be used to reduce toxicity resulting from Cas9 overexpression.

[0336] For ubiquitous expression, the following promoters can be used: CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chain, etc.

[0337] For expression in the brain, the following promoters can be used: synapsin I for all neurons, CaMKIIα for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.

[0338] For expression in the liver, the albumin promoter can be used.

[0339] For expression in the lung, SP-B can be used.

[0340] ICAM can be used on endothelial cells.

[0341] For hematopoietic cells, IFNβ or CD45 can be used.

[0342] OG-2 can be used for osteoblasts.

[0343] Promoters used to drive guide RNAs include: Pol III promoters, e.g., U6 or H1 Expression of gRNA by using a Pol II promoter and intron cassette

[0344] Regarding AAV, AAV can be AAV1, AAV2, AAV5 or any combination thereof. AAV of the AAV related to the target cell can be selected; for example, AAV serotype 1, 2, 5 or hybrid or capsid AAV1, AAV2, AAV5 or any combination thereof can be selected to target brain or nerve cells; and AAV4 can be selected to target cardiac tissue. AAV8 is useful for delivery to the liver. The above promoters and vectors are individually preferred.

[0345] RNA delivery is also a useful in vivo delivery method. Liposomes or nanoparticles can be used to deliver Cas9 and gRNA (and, for example, HR repair template) to cells. Thus, the delivery of CRISPR enzymes, such as Cas9, and / or the delivery of the RNA of the present invention can be in the form of RNA via microvesicles, liposomes, or nanoparticles. For example, Cas9 mRNA and gRNA can be packaged in liposome particles for in vivo delivery. Liposome transfection reagents, such as Life Technologies' Lipofectamine and other commercially available reagents, can effectively deliver RNA molecules to the liver.

[0346] Enhancement of NHEJ or HR efficiency also aids in delivery. NHEJ efficiency is preferably enhanced by co-expressing terminal processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011 August;188(4):787-797). HR efficiency is preferably increased by transiently inhibiting NHEJ machinery such as Ku70 and Ku86. HR efficiency can also be increased by co-expressing prokaryotic or eukaryotic homologous recombination enzymes such as RecBCD and RecA.

[0347] A variety of delivery means are described herein and further discussed in this section.

[0348] Viral delivery: CRISPR enzymes, such as Cas9, and / or any of the RNAs, such as guide RNAs, can be delivered using adeno-associated viruses (AAVs), lentiviruses, adenoviruses, or other viral vector types, or combinations thereof. Cas9 and one or more guide RNAs can be packaged into one or more viral vectors. In some embodiments, the viral vector is delivered to the target tissue, for example, by intramuscular injection, while in other cases, viral delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be via a single dose or multiple doses. Those skilled in the art will understand that the actual dosage delivered herein may vary significantly depending on various factors, such as the choice of vector, the target cell, organism, or tissue, the general condition of the subject, the degree of transformation / modification desired, the route and method of administration, and the type of transformation / modification desired.

[0349] Such dosages may further contain, for example, carriers (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), diluents, pharmaceutically acceptable carriers (e.g., phosphate-buffered saline), pharmaceutically acceptable excipients, adjuvants that enhance antigenicity, immunostimulatory compounds or molecules, and / or other compounds known in the art. Adjuvants herein may include suspensions of minerals (alum, aluminum hydroxide, aluminum phosphate) to which the antigen adsorbs; or water-in-oil emulsions (MF-59, Freund's incomplete adjuvant) in which an antigen solution is emulsified in oil (sometimes containing killed mycobacteria (Freund's complete adjuvant)) to further enhance antigenicity (to inhibit antigen degradation and / or induce macrophage influx). Adjuvants also include immunostimulatory molecules, such as cytokines, costimulatory molecules, and, for example, immunostimulatory DNA or RNA molecules, such as CpG oligonucleotides. Such dosage formulations can be easily ascertained by those skilled in the art. The dosage may further contain one or more pharmaceutically acceptable salts, such as mineral acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and organic acid salts, such as acetate, propionate, malonate, benzoate, and the like. In addition, auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, gels or gelling materials, flavorings, coloring agents, microspheres, polymers, suspending agents, and the like, may also be present herein. In addition, one or more other conventional pharmaceutical ingredients, such as preservatives, humectants, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, and the like, may also be present, especially when the dosage form is a reconstitutable form. Suitable exemplary ingredients include microcrystalline cellulose, sodium carboxymethylcellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin, and combinations thereof.For a thorough discussion of pharmaceutically acceptable excipients, reference may be made to REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991), which is incorporated herein by reference.

[0350] In certain embodiments herein, delivery is via adenovirus, which delivers at least 1 x 10 5 In certain embodiments herein, the dose is preferably at least about 1 x 10 6 particles (e.g., about 1 × 10 6 ~1×10 12 particles), more preferably at least about 1×10 7 particles, more preferably at least about 1×10 8 particles (e.g., about 1 × 10 8 ~1×10 11 particles or approximately 1 x 10 8 ~1×10 12 particles), and most preferably at least about 1×10 0 particles (e.g., about 1 × 10 9 ~1×10 10 particles or approximately 1 x 10 9 ~1×10 12 particles), or even at least about 1 × 10 10 particles (e.g., about 1 × 10 10 ~1×10 12 particles) of adenoviral vector. Alternatively, the dose is about 1 x 10 14 particles or less, preferably about 1 x 10 13 particles or less, and even more preferably about 1×10 12 particles or less, and even more preferably about 1×10 11 particles or less, and most preferably about 1×10 10 particles or less (e.g., about 1 × 10 9 Thus, the dose may be, for example, about 1×10 6 Particle unit (pu), approximately 2 x 10 6 pu, approx. 4×10 6 pu, about 1×107 pu, approx. 2×10 7 pu, approx. 4×10 7 pu, about 1×10 8 pu, approx. 2×10 8 pu, approx. 4×10 8 pu, about 1×10 9 pu, approx. 2×10 9 pu, approx. 4×10 9 pu, about 1×10 10 pu, approx. 2×10 10 pu, approx. 4×10 10 pu, about 1×10 11 pu, approx. 2×10 11 pu, approx. 4×10 11 pu, about 1×10 12 pu, approx. 2×10 12 pu, or approximately 4 × 10 12 The adenovirus vector may contain a single dose of the adenovirus vector, including the adenovirus vector of pu. See, for example, U.S. Patent No. 8,454,972 B2 to Nabel, et al., granted June 4, 2013 (incorporated herein by reference), and the dosage amounts at column 29, lines 36-58 thereof. In some embodiments herein, the adenovirus is delivered in multiple doses.

[0351] In certain embodiments herein, delivery is via AAV. The therapeutically effective dosage for in vivo delivery of AAV to humans is about 1×10 10 ~Approx. 1×10 10 The dosage is expected to be in the range of about 20 to about 50 ml of saline containing 1×10 functional AAV / ml solution. Dosage can be adjusted to balance the therapeutic benefit versus any adverse effects. In certain embodiments herein, the AAV dose is generally about 1×10 5 ~1×10 50 AAV genome, approximately 1 x 10 8 ~1×10 20 AAV genome, approximately 1 x 10 10 ~Approx. 1×10 16 genome, or approximately 1 × 10 11 ~Approx. 1×10 16 The concentration range of AAV genome is approximately 1 x 1013 The AAV may be a genome-wide AAV. Such concentrations can be delivered in about 0.001 ml to about 100 ml, about 0.05 ml to about 50 ml, or about 10 ml to about 25 ml of carrier solution. Other effective dosages can be readily established by those skilled in the art through routine testing to generate dose-response curves. See, for example, U.S. Patent No. 8,404,658 B2 to Hajjar, et al., issued March 26, 2013, column 27, lines 45-60.

[0352] In some embodiments of the present disclosure, delivery is via a plasmid. In such plasmid compositions, the dosage must be a sufficient amount of plasmid to induce a response. For example, a suitable amount of plasmid DNA in a plasmid composition may be about 0.1 to about 2 mg, or about 1 μg to about 10 μg.

[0353] Dosages herein are based on an average 70 kg individual. The frequency of administration is within the discretion of a medical or veterinary practitioner (e.g., physician, veterinarian), or scientist of ordinary skill in the art.

[0354] Lentivirus Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and postmitotic cells. The most commonly known lentivirus is the human immunodeficiency virus (HIV), which uses envelope glycoproteins from other viruses to target a wide range of cell types.

[0355] Lentivirus can be prepared as follows. After cloning of pCasES10 (which contains the lentiviral transfer plasmid backbone), low-passage (p=5) HEK293FT cells were seeded in T-75 flasks to 50% confluence the day before transfection in DMEM containing 10% fetal bovine serum and without antibiotics. After 20 hours, the medium was replaced with OptiMEM (serum-free) medium, and transfection was performed 4 hours later. Cells were transfected with 10 μg of lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (VSV-g pseudotyped), and 7.5 μg of psPAX2 (gag / pol / rev / tat). Transfections were performed in 4 mL of OptiMEM containing cationic lipid delivery agents (50 μL Lipofectamine 2000 and 100 μL Plus Reagent). After 6 hours, the medium was changed to antibiotic-free DMEM containing 10% fetal bovine serum.

[0356] Lentivirus can be purified as follows. After 48 hours, the viral supernatant was collected. The supernatant was first cleared of debris and filtered through a 0.45 μm low protein binding (PVDF) filter. It was then spun in an ultracentrifuge at 24,000 rpm for 2 hours. The viral pellet was resuspended in 50 μl of DMEM overnight at 4°C. It was then divided into aliquots and immediately frozen at -80°C.

[0357] In another embodiment, minimal non-primate lentiviral vectors based on equine infectious anemia virus (EIAV) are also contemplated, particularly for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006;8:275-285, published online November 21, 2005 in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002 / jgm.845). In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector expressing the anti-angiogenic proteins endostain and angiostatin delivered by subretinal injection for the treatment of wet age-related macular degeneration, is also contemplated (see, e.g., Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)), and may be modified for use with the CRISPR-Cas system of the present invention.

[0358] In another embodiment, a self-inactivating lentiviral vector carrying an siRNA targeting a common exon shared by HIV tat / rev, a nucleolus-localizing TAR decoy, and an anti-CCR5 specific hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) can be used and / or adapted for the CRISPR-Cas system of the present invention. A minimum of 2.5 x 10 per kilogram of patient body weight can be used and / or adapted for the CRISPR-Cas system of the present invention. 6 2 x 10 CD34+ cells were harvested. 6 Cells can be pre-stimulated for 16-20 hours in X-VIVO 15 medium (Lonza) containing 2 mM L-glutamine, stem cell factor (100 ng / ml), Flt-3 ligand (Flt-3L) (100 ng / ml), and thrombopoietin (10 ng / ml) (CellGenix) at a density of 100 cells / ml. Pre-stimulated cells are then plated on fibronectin (25 mg / cm). 2 ) (RetroNectin, Takara Bio Inc.) coated 75cm 2Lentivirus can be transduced in tissue culture flasks at a multiplicity of infection of 5 for 16-24 hours.

[0359] Lentiviral vectors have been disclosed for the treatment of Parkinson's disease, for example, see U.S. Patent Application Publication No. 20120295960 and U.S. Patent Nos. 7,303,910 and 7,351,585. Lentiviral vectors have also been disclosed for the treatment of ocular diseases, for example, see U.S. Patent Application Publication Nos. 20060281180, 20090007284, 20110117189; 20090017543; 20070054961, 20100317109. Lentiviral vectors have also been disclosed for delivery to the brain, see, e.g., U.S. Patent Application Publication Nos. 20110293571; 20110293571, 20040013648, 20070025970, 20090111106, and U.S. Patent No. 7,259,015.

[0360] RNA delivery RNA delivery: CRISPR enzymes, such as Cas9, and / or any of the RNAs, such as guide RNA, can also be delivered in the form of RNA. Cas9 mRNA can be produced using in vitro transcription. For example, Cas9 mRNA can be synthesized from a β-globin-polyA tail (a series of 120 or more adenines) using a PCR cassette containing the following elements: T7 promoter-Kozak sequence (GCCACC)-Cas9-3'UTR. This cassette can be used for transcription with T7 polymerase. Guide RNA can also be transcribed using in vitro transcription from a cassette containing a T7 promoter-GG-guide RNA sequence.

[0361] To enhance expression and reduce toxicity, the CRISPR enzyme and / or guide RNA can be modified with pseudo-U or 5-methyl-C.

[0362] mRNA delivery methods currently show particular promise for liver delivery, and in particular AAV8 is particularly preferred for delivery to the liver.

[0363] nanoparticles The CRISPR enzyme mRNA and guide RNA may be delivered simultaneously using nanoparticles or lipid envelopes.

[0364] For example, Su X, Fricke J, Kavanagh DG, and Irvine DJ ("In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles," Mol Pharm. 2011 Jun 6;8(3):774-87. doi:10.1021 / mp100390w. Epub 2011 Apr 1) describe biodegradable core-shell nanoparticles in which a poly(β-amino ester) (PBAE) core is surrounded by a phospholipid bilayer shell. These were developed for in vivo mRNA delivery. The pH-responsive PBAE component was selected to promote endosomal disruption, while the lipid surface layer was selected to minimize the toxicity of the polycation core. Therefore, this is preferred for delivering the RNA of the present invention.

[0365] In one embodiment, nanoparticles based on self-assembling bioadhesive polymers are contemplated, which may be applied to oral, intravenous, and nasal delivery of peptides, all to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs, are also contemplated. Molecular envelope technology involves engineered polymer envelopes that are protected and delivered to disease sites (e.g., Mazza, M. et al. ACS Nano, 2013. 7(2):1016-1026; Siew, A., et al. Mol Pharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012. 161(2):523-36; Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1665-80; Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N.L., et al. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N.L., et al. J Raman Spect, 2012.43(5):681-688; Ahmad, S., et al. J Royal Soc Interface 2010.7:S423-33; Uchegbu, IF Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006.7(12):3452-9 and Uchegbu, IF, et al. Int J Pharm, 2001.224:185-199). Depending on the target tissue, a dose of about 5 mg / kg is contemplated, in single or multiple doses.

[0366] In one embodiment, nanoparticles developed by Dan Anderson's lab at MIT that can deliver RNA to cancer cells to stop tumor growth can be used and / or adapted to the CRISPR Cas system of the present invention. Specifically, the Anderson lab has developed a fully automated combinatorial system for the synthesis, purification, characterization, and formulation of novel biomaterials and nanoformulations. For example, Alabi et al.,Proc Natl Acad Sci US A.2013 Aug 6;110(32):12881-6;Zhang et al.,Adv Mater.2013 Sep 6;25(33):4641-5;Jiang et al.,Nano Lett.2013 Mar 13;13(3):1059-64;Karagiannis et al. See Whitehead et al., ACS Nano.2012 Aug 28;6(8):6922-9 and Lee et al., Nat Nanotechnol.2012 Jun 3;7(6):389-93.

[0367] US Patent Application Publication No. 20110293703 relates to lipidoid compounds that are particularly useful for administering polynucleotides, which can be applied to the delivery of the CRISPR Cas system of the present invention. In one embodiment, an aminoalcohol lipidoid compound is combined with a drug to be delivered to a cell or subject to form a microparticle, nanoparticle, liposome, or micelle. The drug delivered by the particle, liposome, or micelle may be in gas, liquid, or solid form, and the drug may be a polynucleotide, protein, peptide, or small molecule. The aminoalcohol lipidoid compound may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form particles. These particles may then optionally be combined with pharmaceutical excipients to form pharmaceutical compositions.

[0368] US Patent Publication No. 0110293703 also provides a method for preparing aminoalcohol lipidoid compounds. The aminoalcohol lipidoid compounds of the present invention are formed by reacting one or more equivalents of an amine with one or more equivalents of an epoxide-terminated compound under suitable conditions. In certain embodiments, all amino groups on the amine react completely with the epoxide-terminated compound to form a tertiary amine. In other embodiments, not all amino groups on the amine react completely with the epoxide-terminated compound to form a tertiary amine, thus resulting in primary or secondary amines in the aminoalcohol lipidoid compound. These primary or secondary amines can remain or can be reacted with another electrophile, such as a different epoxide-terminated compound. As will be understood by those skilled in the art, reacting an amine with less than an excess of an epoxide-terminated compound can result in multiple different aminoalcohol lipidoid compounds with various numbers of tails. Certain amines can be fully functionalized with two epoxide-derived compound tails, while other molecules are not fully functionalized with epoxide-derived compound tails. For example, a diamine or polyamine may contain one, two, three, or four epoxide-derived compound tails on various amino moieties of the molecule, resulting in primary, secondary, and tertiary amines. In certain embodiments, not all amino groups are fully functionalized. In certain embodiments, two of the same type of epoxide-terminated compound are used. In other embodiments, two or more different epoxide-terminated compounds are used. The synthesis of amino alcohol lipidoid compounds may be carried out with or without solvent, and the synthesis may be carried out at elevated temperatures ranging from 30 to 100°C, preferably about 50 to 90°C. The prepared amino alcohol lipidoid compounds may optionally be purified. For example, a mixture of amino alcohol lipidoid compounds may be purified to produce an amino alcohol lipidoid compound with a specific number of epoxide-derived compound tails, or a mixture may be purified to produce a specific stereoisomer or positional isomer. The amino alcohol lipidoid compounds may also be alkylated and / or acylated using alkyl halides (e.g., methyl iodide) or other alkylating agents.

[0369] U.S. Patent Application Publication No. 0110293703 also provides libraries of amino alcohol lipidoid compounds prepared by the methods of the invention. These amino alcohol lipidoid compounds can be prepared and / or screened using high-throughput techniques involving liquid handlers, robots, microtiter plates, computers, etc. In certain embodiments, amino alcohol lipidoid compounds are screened for their ability to transfect polynucleotides or other agents (e.g., proteins, peptides, small molecules) into cells.

[0370] U.S. Patent Application Publication No. 20130302401 describes a class of poly(β-amino alcohols) (PBAAs) prepared using combinatorial polymerization. The PBAAs of the present invention can be used in biotechnological and biomedical applications as coatings (such as thin or multilayer coatings for medical devices or implants), additives, materials, excipients, non-biofouling agents, micropatterning agents, and cell encapsulation agents. When used as surface coatings, these PBAAs induced varying levels of inflammation both in vitro and in vivo depending on their chemical structure. The large chemical diversity of this material class made it possible to identify polymer coatings that inhibit macrophage activation in vitro. Furthermore, these coatings reduced inflammatory cell recruitment and fibrosis after subcutaneous implantation of carboxylated polystyrene microparticles. These polymers can be used to form polyelectrolyte complex capsules for cell encapsulation. This invention may also have many other biological applications, such as antimicrobial coatings, DNA or siRNA delivery, and stem cell tissue engineering. The teachings of US Patent Application Publication No. 20130302401 may be applied to the CRISPR Cas system of the present invention.

[0371] In another embodiment, lipid nanoparticles (LNPs) are contemplated. Specifically, anti-transthyretin small interfering RNA encapsulated in lipid nanoparticles (see, e.g., Coelho et al., N Engl J Med 2013;369:819-29) may be applied to the CRISPR-Cas system of the present invention. Doses of about 0.01 to about 1 mg / kg body weight administered intravenously are contemplated. Medication to reduce the risk of injection-related reactions is contemplated, including dexamethasone, acetaminophen, diphenhydramine or cetirizine, and ranitidine. Multiple doses of about 0.3 mg per kilogram every four weeks for five doses are also contemplated.

[0372] LNP has been shown to be highly effective in delivering siRNA to the liver (see, for example, Tabernero et al., Cancer Discovery, April 2013, Vol. 3, No. 4, pp. 363-470), and therefore delivery of CRISPR Cas to the liver is contemplated. A dosage of approximately four doses of 6 mg / kg LNP every two weeks may be contemplated. [Tabernero et al.] demonstrated that tumor regression was observed after the first two cycles of LNP administered at 0.7 mg / kg, and by the end of six cycles, the patient achieved a partial response, with complete regression of lymph node metastases and substantial shrinkage of the liver tumor. This patient achieved a complete response after 40 doses, and the patient remained in remission after 26 months of receiving doses, completing treatment. The patient with RCC and two patients with extrahepatic disease sites, including kidney, lung, and lymph node disease, who had progressed after previous treatment with a VEGF pathway inhibitor, remained stable at all sites for approximately 8 to 12 months, and the patient with PNET and liver metastases continued on the extension study for 18 months (36 doses) with stable disease.

[0373] However, the charge of LNPs must be considered. Cationic lipids, when combined with negatively charged lipids, induce nonbilayer structures, thereby facilitating intracellular delivery. Because charged LNPs are rapidly cleared from the circulation after intravenous injection, ionizable cationic lipids with pKa values ​​below 7 have been developed (see, e.g., Rosin et al., Molecular Therapy, vol. 19, no. 12, pp. 1286-2200, December 2011). Negatively charged polymers, such as siRNA oligonucleotides, can be loaded into LNPs at low pH values ​​(e.g., pH 4), where the ionizable lipids exhibit a positive charge. However, at physiological pH values, LNPs exhibit a low surface charge, which is compatible with longer circulation times. Four ionizable cationic lipids have received attention: 1,2-dilineoyl-3-dimethylammonium propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). These lipid-containing LNP siRNA systems have been shown to exhibit significantly different gene silencing properties in hepatocytes in vivo, with different potencies in the order DLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP using a factor VII gene silencing model (see, for example, Rosin et al., Molecular Therapy, vol. 19, no. 12, pp. 1286-2200, December 2011). In particular, dosages of 1 μg / ml level can be contemplated for formulations containing DLinKC2-DMA.

[0374] Preparation of LNPs and CRISPR Cas inclusion according to Rosin et al., Molecular Therapy, vol. 19, no. 12, pp. 1286-2200, December 2011) can be used and / or adapted. The cationic lipids 1,2-diline...

Claims

1. A non-naturally occurring or engineered composition comprising a guide RNA (sgRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein at least one loop of the sgRNA is modified with an insertion of a distinct RNA sequence that binds to one or more adaptor proteins, wherein the adaptor protein is associated with one or more functional domains.

2. A non-naturally occurring or engineered CRISPR-Cas complex composition comprising the sgRNA of claim 1 and a CRISPR enzyme.

3. the CRISPR enzyme comprises at least one mutation such that the CRISPR enzyme has 5% or less of the nuclease activity of a CRISPR enzyme that does not have the at least one mutation; and / or Contains at least one nuclear localization sequence, 3. The non-naturally occurring or engineered CRISPR-Cas complex composition of claim 2.

4. 4. The sgRNA of claim 1 or the CRISPR-Cas complex of claim 2 or 3, wherein one or more adaptor proteins associated with one or more functional domains are present and bind to distinct RNA molecules inserted into at least one loop of the sgRNA.

5. A non-naturally occurring or engineered composition, comprising: one or more guide RNAs (sgRNAs) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell; CRISPR enzyme comprising at least one nuclear localization sequence Including, the CRISPR enzyme comprises at least one mutation such that the CRISPR enzyme has 5% or less of the nuclease activity of a CRISPR enzyme that does not have the at least one mutation; at least one loop of at least one sgRNA is modified with an insertion of a distinct RNA sequence that binds to one or more adaptor proteins; The adaptor protein is associated with one or more functional domains. composition.

6. 6. The composition of any one of claims 2, 3, 4 or 5, wherein the CRISPR enzyme has a nuclease activity that is at least 97%, or 100%, reduced compared to a CRISPR enzyme that does not have the at least one mutation.

7. 7. The composition of any one of claims 2, 3, 4, 5 or 6, wherein the CRISPR enzyme comprises two or more mutations, where two or more of D10, E762, H840, N854, N863, or D986 by the SpCas9 protein or any corresponding ortholog, or N580 by the SaCas9 protein, are mutated, or wherein the CRISPR enzyme comprises at least one mutation, where at least one of H840 is mutated.

8. 8. The composition of claim 7, wherein the CRISPR enzyme comprises two or more mutations including D10A, E762A, H840A, N854A, N863A or D986A by an SpCas9 protein or any corresponding ortholog, or N580A by a SaCas9 protein, or at least one mutation including H840A.

9. 7. The composition of any one of claims 2, 3, 4, 5 or 6, wherein the CRISPR enzyme comprises H840A, or D10A and H840A, or D10A and N863A, from an SpCas9 protein or any corresponding ortholog.

10. The CRISPR enzyme is N580A by the SaCas9 protein or any corresponding ortholog; or D10A by the SpCas9 protein or any corresponding ortholog, and N580A by the SaCas9 protein 7. The composition of claim 2, 3, 4, 5 or 6, comprising:

11. The composition of any one of claims 2 to 10, wherein the CRISPR enzyme is associated with one or more functional domains.

12. The composition of claim 11 , wherein the one or more functional domains associated with the adaptor protein are heterologous functional domains.

13. 12. The composition of claim 11, wherein the one or more functional domains associated with the CRISPR enzyme are heterologous functional domains.

14. 14. The composition of any one of claims 1 to 13, wherein the adaptor protein is a fusion protein comprising the functional domain, the fusion protein optionally comprising a linker between the adaptor protein and the functional domain, the linker optionally comprising a GlySer linker.

15. 15. The composition of any one of claims 1-14, wherein at least one loop of the sgRNA is unmodified with an insertion of a distinct RNA sequence that binds to one or more adaptor proteins, and optionally one of the unmodified sgRNA loops is one of a tetraloop or stem loop 2.

16. The composition of any one of claims 1 to 15, wherein the one or more functional domains associated with the adaptor protein are transcription activation domains.

17. The composition of any one of claims 11 to 16, wherein the one or more functional domains associated with the CRISPR enzyme are transcription activation domains.

18. 18. The composition of any one of claims 1 to 17, wherein the one or more functional domains associated with the adaptor protein are transcription activation domains including VP64, p65, MyoD1, HSF1, RTA or SET7 / 9.

19. 19. The composition of any one of claims 11 to 18, wherein the one or more functional domains associated with the CRISPR enzyme are transcription activation domains including VP64, p65, MyoD1, HSF1, RTA or SET7 / 9.

20. The composition of any one of claims 1 to 15, wherein the one or more functional domains associated with the adaptor protein are transcriptional repressor domains.

21. The composition of any one of claims 11 to 16, wherein the one or more functional domains associated with the CRISPR enzyme are transcriptional repressor domains.

22. 22. The composition of claim 20 or 21, wherein the transcriptional repressor domain is a KRAB domain.

23. 22. The composition of claim 20 or 21, wherein the transcriptional repressor domain is a NuE domain, an NcoR domain, a SID domain, or a SID4X domain.

24. 16. The composition of any one of claims 1 to 15, wherein the one or more functional domains associated with the adaptor protein have one or more activities including methylase activity, demethylase activity, transcription activating activity, transcription repressing activity, transcription release factor activity, histone modifying activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity or nucleic acid binding activity.

25. 16. The composition of any one of claims 11 to 15, wherein the one or more functional domains associated with the CRISPR enzyme have one or more activities including methylase activity, demethylase activity, transcriptional activator activity, transcriptional repressor activity, transcriptional release factor activity, histone modifying activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, nucleic acid binding activity, or molecular switch activity or chemically or light inducible.

26. The composition according to any one of claims 24 to 25, wherein the DNA cleavage activity is due to a nuclease.

27. 27. The composition of claim 26, wherein the nuclease comprises a Fok1 nuclease.

28. 27. The composition of any one of claims 2-26, wherein the one or more functional domains are attached to the CRISPR enzyme such that, upon binding to the sgRNA and target, the functional domains are in a spatial orientation that allows the functional domains to function in their attributed function.

29. 30. The composition of claim 28, wherein the one or more functional domains are attached to the CRISPR enzyme via a linker, optionally a GlySer linker.

30. 30. The composition of any one of claims 2-29, wherein the sgRNA is modified such that, after sgRNA binds to the adaptor protein, and further binds to the CRISPR enzyme and target, the functional domain is in a spatial orientation that allows the functional domain to function in its attributed function.

31. 28. The composition of any one of claims 3 or 5-27, wherein the at least one loop of the sgRNA is a tetraloop and / or loop 2.

32. 32. The composition of Claim 31, wherein the tetraloop and loop 2 of the sgRNA are modified with an insertion of the distinct RNA sequence.

33. 33. The composition of any one of claims 31 or 32, wherein the insert of distinct RNA sequence that binds to one or more adaptor proteins is an aptamer sequence.

34. The composition of claim 33, wherein the aptamer sequences are two or more aptamer sequences specific for the same adaptor protein.

35. 34. The composition of claim 33, wherein the aptamer sequence is two or more aptamer sequences specific for different adaptor proteins.

36. The composition of any one of claims 1 to 35, wherein the adaptor protein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, or PRR1.

37. The composition according to any one of claims 1 to 36, wherein the cell is a eukaryotic cell, optionally a mammalian cell.

38. 38. The composition of claim 37, wherein the cell is a human cell or a mouse cell.

39. 39. A mammalian cell according to claim 37 or 38, comprising a cell line, optionally a human cell line or a murine cell line.

40. 39. A transgenic mammal model, optionally a mouse, transformed with a composition according to claim 37 or 38 or a descendant of said transformant.

41. 37. A method of introducing a genomic locus event comprising administration to a host, or expression in said host, of one or more compositions of any one of claims 1 to 36.

42. 42. The method of claim 41, wherein the genomic locus event comprises affecting gene activation, gene inhibition, or a cleavage or insertion of DNA in the locus.

43. 43. The method of claim 41 or 42, comprising delivery of the composition or a nucleic acid molecule encoding same, said nucleic acid molecule being operably linked to a regulatory sequence and expressed in vivo.

44. 43. The method of claim 42, wherein the in vivo expression is via lentivirus, adenovirus, or AAV.

45. A vector comprising a nucleic acid molecule encoding a guide RNA (sgRNA), the vector comprising a regulatory element operable in a eukaryotic cell comprising a guide sequence (sgRNA) operably linked to a promoter, the sgRNA capable of hybridizing to a target sequence in a genomic locus of interest in the cell, and at least one loop of the sgRNA being modified with the insertion of a distinct RNA sequence that binds to one or more adaptor proteins.

46. 1. A vector comprising a regulatory element operable in a eukaryotic cell comprising a nucleic acid molecule encoding a CRISPR enzyme operably linked to a promoter, the enzyme comprising at least one nuclear localization sequence, the CRISPR enzyme comprising at least one mutation such that the CRISPR enzyme has 5% or less of the nuclease activity of a CRISPR enzyme not having said at least one mutation, and the CRISPR enzyme is associated with one or more functional domains.

47. A nucleic acid molecule encoding the sgRNA of claim 1 or the CRISPR-Cas complex of claim 2 or 3 or the composition of claim 5.

48. 41. A method of screening for gain of function (GOF) or loss of function (LOF), comprising cells of a cell line of claim 39 or a model of claim 40 that contain or express Cas9, the method comprising introducing the composition of claim 1 into cells of the cell line or model, wherein the sgRNA comprises either an activator or a repressor, and monitoring GOF or LOF, respectively, for those cells in which the introduced sgRNA comprises an activator or for those cells in which the introduced sgRNA comprises a repressor.

49. The composition of any one of claims 2 to 15, wherein the CRISPR enzyme comprises one or more functional domains.

50. 50. The composition of any one of claims 5-38 or 49, wherein there is multiplexing when two or more sgRNAs are present and the sgRNAs target different sequences and are thereby used.

51. 51. The composition of claim 50, wherein there are two or more sgRNAs modified with insertions of distinct RNA sequences that bind to one or more adaptor proteins.

52. 52. The composition of claim 50 or 51, wherein one or more adaptor proteins associated with one or more functional domains are present and bind to the distinct RNA sequence inserted into at least one loop of the sgRNA.

53. A CRISPR Cas complex comprising a CRISPR enzyme and a guide RNA (sgRNA), the CRISPR enzyme comprises at least one mutation such that the CRISPR enzyme has 5% or less of the nuclease activity of a CRISPR enzyme that does not have the at least one mutation, and optionally at least one or more nuclear localization sequences; The guide RNA (sgRNA) comprises a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell; a) the CRISPR enzyme is associated with two or more functional domains, which may be the same or different; or b) at least one loop of the sgRNA is modified with the insertion of a distinct RNA sequence that binds to one or more adaptor proteins, the adaptor proteins being associated with two or more functional domains; or c) a complex wherein the CRISPR enzyme is associated with one or more functional domains, and at least one loop of the sgRNA is modified with the insertion of a distinct RNA sequence that binds to one or more adaptor proteins, the adaptor proteins being associated with one or more functional domains.

54. The composition of any one of claims 5 to 38 or 49 to 52, wherein the target sequence is a non-coding or regulatory sequence.

55. The composition of claim 54, wherein the regulatory sequence is a promoter, enhancer or silencer sequence.

56. (A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors, The vector comprises: I. A first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, said polynucleotide sequence comprising: (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) a tracr mate sequence, and (c) tracr sequence A first regulatory element comprising: II. A second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme comprising at least one nuclear localization sequence. Including, (a), (b) and (c) are arranged in a 5' to 3' orientation; Components I and II are located on the same or different vectors of the system, the tracr mate sequence, when transcribed, hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence; The CRISPR complex comprises: (1) the guide sequence hybridized to the target sequence; and (2) the CRISPR enzyme complexed with the tracr mate sequence hybridized to the tracr sequence. or (B) A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors, said vectors comprising: I. (a) a guide sequence capable of hybridizing to a target sequence in a prokaryotic cell, and (b) at least one tracr mate sequence; A first regulatory element operably linked to II. A second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme; and III. A third regulatory element operably linked to the tracr sequence Including, Components I, II and III are located on the same or different vectors of said system, the tracr mate sequence, when transcribed, hybridizes to the tracr sequence, and the guide sequence directs sequence-specific target binding of a CRISPR complex to the target sequence; The CRISPR complex comprises: (1) the guide sequence hybridized to the target sequence; and (2) the CRISPR enzyme complexed with the tracr mate sequence hybridized to the tracr sequence; The CRISPR enzyme is selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, and the like. orthologues of Cas9 from genera belonging to the group consisting of: Bacillus subtilis, ... The Cas comprises a helical domain 2 truncation.

57. 57. The composition of claim 56, wherein the CRISPR enzyme has one or more mutations in any domain of the enzyme.

58. 58. The composition of claim 56 or 57, wherein the CRISPR enzyme has one or more mutations in the catalytic domain.

59. 58. The composition of claim 56 or 57, wherein the one or more mutations are present in the RuvCI, RuvCII, RuvCIII or HNH domain of the CRISPR enzyme.

60. 60. The composition of claim 59, wherein the CRISPR enzyme comprises one or more mutations corresponding to the position numbering of SpCas9 or any corresponding ortholog at positions D10A, E762A, D986A, S15A, E762A, H982A, H983A, D986A, H840A, N854A or N863A, or N580 is mutated by the SaCas9 protein.

61. 57. The composition of claim 56, wherein the enzyme further comprises a functional domain.

62. 62. The composition of claim 61, wherein the functional domain is a transcriptional activator domain.

63. 63. The composition of claim 62, wherein the transcriptional activator domain is VP64.

64. 62. The composition of claim 61, wherein the functional domain is a transcriptional repressor domain.

65. 65. The composition of claim 64, wherein the transcriptional repressor domain is KRAB, SID or SID4X.

66. 57. The composition of claim 56, wherein the Cas9 ortholog is Sutterella wadsworthensis Cas9.

67. 57. The composition of claim 56, wherein the Cas9 ortholog is Filifactor alocis Cas9.

68. 57. The composition of claim 56, wherein the Cas9 ortholog is Lactobacillus johnsonii Cas9.

69. 57. The composition of claim 56, wherein the Cas9 ortholog is Campylobacter lari Cas9.

70. 57. The composition of claim 56, wherein the Cas9 ortholog is Corynebacter diptheriae Cas9.

71. 57. The composition of claim 56, wherein the Cas9 ortholog is Parvibaculum lavamentivorans Cas9.

72. 57. The composition of claim 56, wherein the Cas9 ortholog is Mycoplasma gallisepticum Cas9.

73. 57. The composition of claim 56, wherein the Cas9 ortholog is Staphylococcus aureus subspecies Aureus Cas9.

74. 57. The composition of claim 56, wherein the Cas9 ortholog is Legionella pneumophila Paris Cas9.

75. 57. The composition of claim 56, wherein the Cas9 ortholog is Treponema denticola Cas9.

76. 57. The composition of claim 56, wherein the Cas9 ortholog is Staphylococcus pseudointermedius Cas9.

77. 57. The composition of claim 56, wherein the Cas9 ortholog is Neisseria cinerea Cas9.

78. 1. A method for modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest, comprising: Delivering a non-naturally occurring or engineered composition comprising a vector system operably encoding the composition of claim 56 for expression thereof. The method includes:

79. 79. The method of claim 78, wherein delivery is via one or more viral vectors.

80. A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors, The vector comprises: I. A first regulatory element operably linked to a first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, said first polynucleotide sequence comprising: (i) a first guide sequence capable of hybridizing to a first target sequence at a first genomic locus in a cell of the organism; (ii) a first tracr mate sequence, and (iii) a first tracr sequence A first regulatory element comprising: II. A second regulatory element operably linked to a second CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, said second polynucleotide sequence comprising: (i) a second guide sequence capable of hybridizing to a second target sequence at a second genomic locus in a cell of the organism; (ii) a second tracr mate sequence, and (iii) a second tracr sequence A second regulatory element comprising: III. A third regulatory element operably linked to an enzyme coding sequence encoding a first CRISPR enzyme comprising at least one or more nuclear localization sequences and operably linked to a first functional domain; IV. A fourth regulatory element operably linked to an enzyme coding sequence encoding a second CRISPR enzyme comprising at least one nuclear localization sequence and operably linked to a second functional domain. Including, (i), (ii) and (iii) of I and II are arranged in a 5' to 3' orientation; Components I, II, III and IV are located on the same or different vectors of said system, each tracr mate sequence, when transcribed, hybridizes to its corresponding tracr sequence, and the first and second guide sequences direct sequence-specific binding of the first and second CRISPR complexes to the first and second target sequences; the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence, wherein expression of the CRISPR enzyme provides for manipulation of the target sequence; wherein the first and second CRISPR enzymes each comprise two or more mutations; The first and second CRISPR enzymes are selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, and the like. acterium), Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter, The Cas comprises a helical domain 2 truncation.

81. The composition of claim 80, wherein the first functional domain is selected from the group consisting of a transcriptional activator, a transcriptional repressor, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, a cryptochrome, and a light-inducible / regulatory domain or a chemically-inducible / regulatory domain.

82. The composition of claim 80, wherein the second functional domain is selected from the group consisting of a transcriptional activator, a transcriptional repressor, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, a cryptochrome, and a light-inducible / regulatory domain or a chemically-inducible / regulatory domain.

83. 81. The composition of claim 80, wherein the first or second functional domain is a transcriptional activator domain.

84. 84. The composition of claim 83, wherein the transcriptional activator domain is VP64.

85. 81. The composition of claim 80, wherein the first or second functional domain is a transcriptional repressor domain.

86. 86. The composition of claim 85, wherein the transcriptional repressor domain is KRAB, SID or SID4X.

87. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Sutterella wadsworthensis Cas9.

88. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Filifactor alocis Cas9.

89. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Lactobacillus johnsonii Cas9.

90. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Campylobacter lari Cas9.

91. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Corynebacter diptheriae Cas9.

92. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Parvibaculum lavamentivorans Cas9.

93. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Mycoplasma gallisepticum Cas9.

94. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Staphylococcus aureus subsubspecies Aureus Cas9.

95. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Legionella pneumophila Paris Cas9.

96. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Treponema denticola Cas9.

97. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Staphylococcus pseudointermedius Cas9.

98. 81. The composition of claim 80, wherein the first or second CRISPR enzyme is Neisseria cinerea Cas9.

99. 1. A method for modulating expression of two or more genomic loci of interest in an organism, comprising: Delivering non-naturally occurring or engineered compositions comprising vector systems comprising one or more vectors and the vector comprises I. A first regulatory element operably linked to a first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, said first polynucleotide sequence comprising: (i) a first guide sequence capable of hybridizing to a first target sequence at a first genomic locus in a cell of the organism; (ii) a first tracr mate sequence, and (iii) a first tracr sequence a first regulatory element comprising: II. A second regulatory element operably linked to a second CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, said second polynucleotide sequence comprising: (i) a second guide sequence capable of hybridizing to a second target sequence at a second genomic locus in a cell of the organism; (ii) a second tracr mate sequence, and (iii) a second tracr sequence A second regulatory element comprising: III. A third regulatory element operably linked to an enzyme coding sequence encoding a first CRISPR enzyme comprising at least one or more nuclear localization sequences and operably linked to a first functional domain; IV. A fourth regulatory element operably linked to an enzyme coding sequence encoding a second CRISPR enzyme comprising at least one nuclear localization sequence and operably linked to a second functional domain. Including, (i), (ii) and (iii) of I and II are arranged in a 5' to 3' orientation; Components I, II, III and IV are located on the same or different vectors of said system, each tracr mate sequence, when transcribed, hybridizes to its corresponding tracr sequence, and the first and second guide sequences direct sequence-specific binding of the first and second CRISPR complexes to the first and second target sequences; the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence, wherein expression of the CRISPR enzyme provides for manipulation of the target sequence; wherein the first and second CRISPR enzymes each comprise two or more mutations; The first and second CRISPR enzymes are selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, and the like. acterium), Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter, The Cas comprises a helical domain 2 truncation, The first genomic locus is modulated by the activity of the first functional domain and the second genomic locus is modulated by the activity of the second functional domain.

100. The method of claim 99, wherein the first functional domain is selected from the group consisting of a transcriptional activator, a transcriptional repressor, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, a cryptochrome, and a light-inducible / regulatory domain or a chemically-inducible / regulatory domain.

101. The method of claim 99, wherein the second functional domain is selected from the group consisting of a transcriptional activator, a transcriptional repressor, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, a cryptochrome, and a light-inducible / regulatory domain or a chemically-inducible / regulatory domain.

102. 100. The method of claim 99, wherein the first or second functional domain is a transcriptional activator domain.

103. The method of claim 102, wherein the transcriptional activator domain is VP64.

104. 100. The method of claim 99, wherein the first or second functional domain is a transcriptional repressor domain.

105. The method of claim 104, wherein the transcriptional repressor domain is KRAB, SID or SID4X.

106. 100. The method of claim 99, wherein the first or second CRISPR enzyme is Sutterella wadsworthensis Cas9.

107. 100. The method of claim 99, wherein the first or second CRISPR enzyme is Filifactor alocis Cas9.

108. 100. The method of claim 99, wherein the first or second CRISPR enzyme is Lactobacillus johnsonii Cas9.

109. 100. The composition of claim 99, wherein the first or second CRISPR enzyme is Campylobacter lari Cas9.

110. 71. The method of claim 70, wherein the first or second CRISPR enzyme is Corynebacter diptheriae Cas9.

111. 100. The method of claim 99, wherein the first or second CRISPR enzyme is Parvibaculum lavamentivorans Cas9.

112. 100. The method of claim 99, wherein the first or second CRISPR enzyme is Mycoplasma gallisepticum Cas9.

113. 100. The method of claim 99, wherein the first or second CRISPR enzyme is Staphylococcus aureus subsubspecies Aureus Cas9.

114. 100. The method of claim 99, wherein the first or second CRISPR enzyme is Legionella pneumophila Paris Cas9.

115. 100. The method of claim 99, wherein the first or second CRISPR enzyme is Treponema denticola Cas9.

116. 100. The method of claim 99, wherein the first or second CRISPR enzyme is Staphylococcus pseudointermedius Cas9.

117. 100. The method of claim 99, wherein the first or second CRISPR enzyme is Neisseria cinerea Cas9.

118. 1. A method of treating or inhibiting a pathology caused by a deletion of a target sequence in a genomic locus of interest in a subject or non-human subject in need thereof, the method comprising modifying the subject or non-human subject by manipulation of said target sequence, the pathology being: delivering a non-naturally occurring or engineered composition comprising an AAV vector system comprising one or more AAV vectors operably encoding the composition of claim 56 for expression thereof, wherein said composition manipulates said target sequence when expressed. The method of claim 1, wherein the target sequence is susceptible to treatment or inhibition by manipulation of the target sequence comprising providing a treatment comprising:

119. 119. The method of claim 118, comprising inducing expression.

120. 119. The method of claim 118, wherein the organism or subject is a eukaryote or a non-human eukaryote.

121. 119. The method of claim 118, wherein the organism or subject is a plant.

122. 119. The method of claim 118, wherein the organism or subject is a mammal or a non-human mammal.

123. 119. The method of claim 118, wherein the organism or subject is algae.

124. The method of claim 118, wherein the viral vector is AAV.

125. A modified CRISPR enzyme, comprising: Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaete, and the like; orthologues of Cas9 from genera belonging to the group consisting of Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter, and differences from the corresponding wild-type CRISPR enzyme include: the modified CRISPR enzyme is truncated compared to a wild-type CRISPR enzyme; or and / or the CRISPR enzyme has a length of at least 500 amino acids, at least 800-899 amino acids, at least 900-999 amino acids, at least 1000-1099 amino acids, at least 1100-1199 amino acids, at least 1200-1299 amino acids, at least 1300-1399 amino acids, at least 1400-1499 amino acids, at least 1500-1599 amino acids, at least 1600-1699 amino acids or at least 2000 amino acids; and / or the CRISPR enzyme is a nuclease that directs cleavage of both strands at the location of the target sequence, or the CRISPR enzyme is a nickase that directs cleavage of one strand at the location of the target sequence, and / or the CRISPR enzyme is codon-optimized or codon-optimized for expression in a eukaryotic cell, and / or the CRISPR enzyme comprises one or more mutations, the CRISPR enzyme comprises a chimeric CRISPR enzyme; and / or The Cas comprises a helical domain 2 truncation Modified CRISPR enzyme.

126. 71. The modified CRISPR enzyme of claim 70, or the composition of claim 56, wherein the CRISPR enzyme has one or more mutations in any domain of the enzyme.

127. 126. The modified CRISPR enzyme of claim 125, having one or more mutations in the catalytic domain.

128. 126. The modified CRISPR enzyme of Claim 125, wherein said one or more mutations are present in a RuvCI, RuvCII, RuvCIII or HNH domain of said CRISPR enzyme.

129. 126. The modified CRISPR enzyme of claim 125, comprising one or more mutations corresponding to the position numbering of SpCas9 or any corresponding ortholog at positions D10A, E762A, D986A, S15A, E762A, H982A, H983A, D986A, H840A, N854A or N863A, or N580 is mutated by the SaCas9 protein.

130. 57. The modified CRISPR enzyme of claim 56, further comprising a functional domain.

131. 126. The modified CRISPR enzyme of claim 125, wherein the functional domain is a transcriptional activator domain.

132. 127. The modified CRISPR enzyme of claim 126, wherein the transcription activator domain is VP64.

133. 126. The modified CRISPR enzyme of claim 125, wherein the functional domain is a transcriptional repressor domain.

134. 134. The modified CRISPR enzyme of claim 133, wherein the transcriptional repressor domain is KRAB, SID, or SID4X.

135. 136. A composition according to any one of claims 56 to 77 or 80 to 98 or a CRISPR enzyme according to any one of claims 125 to 134 for use in medicine.

136. A composition according to any one of claims 56 to 77 or 80 to 98 or a CRISPR enzyme according to any one of claims 125 to 134, for use in a method according to any one of claims 78, 79, 99 to 134.

137. 136. Use of the composition of any one of claims 56-77 or 80-98 or the CRISPR enzyme of any one of claims 125-134 in ex vivo gene or genome editing.

138. Use of a composition according to any one of claims 56 to 77 or 80 to 98 or a CRISPR enzyme according to any one of claims 125 to 134 in the manufacture of a medicament for ex vivo gene or genome editing, or in a method according to any one of claims 78, 79, 99 to 134.

139. 135. The composition of any one of claims 56-77 or 80-983 or the CRISPR enzyme of any one of claims 125-134, wherein the target sequence is flanked at its 3' end by 5'-NRG (wherein N is any nucleotide), particularly when the Cas9 is (or is derived from) a S. pyogenes or S. aureus Cas9.

140. A method for preparing an AAV for delivery of the composition described in claim 56 or 80 or the modified enzyme described in claim 125, comprising transfecting a plasmid containing or essentially consisting of a nucleic acid molecule encoding an AAV into an AAV-infected cell, and supplying the AAV rep and / or cap essential for replication and packaging of the AAV.

141. The method of claim 140, wherein the cells are transfected with a helper plasmid or helper virus to provide the AAV rep and / or cap necessary for replication and packaging of the AAV.

142. 142. The method of claim 141, wherein the helper virus is a poxvirus, adenovirus, herpesvirus or baculovirus.

143. 143. The method of claim 142, wherein the poxvirus is a cowpox virus.

144. - the cell is a mammalian cell; or - the cell is an insect cell and the helper virus is a baculovirus, The method of claim 140.

145. 126. A method of delivering a CRISPR enzyme according to claim 125, comprising delivering to a cell an mRNA encoding said CRISPR enzyme.

146. A chimeric Cas enzyme comprising one or more fragments from a first Cas ortholog and one or more fragments from a second Cas ortholog; The Cas is an enzyme containing a helical domain 2 truncation.

147. 147. The enzyme of claim 146, wherein the one or more fragments from the first or second Cas ortholog are from the C- or N-terminus of the first or second Cas ortholog.

148. The first or second Cas ortholog is selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, and the like.

147. The enzyme of claim 146, wherein the genera are selected from the group consisting of: Bacillus subtilis, ...

149. Use of a composition or an enzyme according to any one of claims 1 to 38, 49 to 52, 54 to 77, 80 to 98, 125 to 136, 139 and 146 to 148 in the preparation of a medicament for the modification of a target sequence.

150. 57. The composition of claim 56, wherein the CRISPR enzyme is truncated compared to a wild-type CRISPR enzyme, or wherein the CRISPR enzyme comprises between 500 and 2000 amino acids.

151. 57. The composition of claim 56, wherein the CRISPR enzyme is a nuclease that directs cleavage of both strands at the location of the target sequence, or wherein the CRISPR enzyme is a nickase that directs cleavage of one strand at the location of the target sequence.

152. 57. The composition of claim 56, wherein the guide sequence comprises at least 15 nucleotides.

153. 57. The composition of claim 56, wherein the CRISPR enzyme is codon optimized or is codon optimized for expression in a eukaryotic cell.

154. 57. The composition of claim 56, wherein the CRISPR enzyme comprises one or more mutations.

155. 57. The composition of claim 56, wherein the CRISPR enzyme comprises a chimeric CRISPR enzyme.

156. A non-naturally occurring or engineered composition, comprising: A)-I. A CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) a tracr mate sequence, and (c) tracr sequence A polynucleotide sequence comprising: II. Polynucleotide sequence encoding a CRISPR enzyme comprising at least one nuclear localization sequence Including, (a), (b) and (c) are arranged in a 5' to 3' orientation; the tracr mate sequence, when transcribed, hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence; the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence, and the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA; or (B) I. (a) a guide sequence capable of hybridizing to a target sequence in a prokaryotic cell, and (b) at least one tracr mate sequence; A polynucleotide comprising II. A polynucleotide sequence encoding a CRISPR enzyme, and III. Polynucleotide sequences containing tracr sequences Including, the tracr mate sequence, when transcribed, hybridizes to the tracr sequence, and the guide sequence directs sequence-specific target binding of a CRISPR complex to the target sequence; the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence, and the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA; The CRISPR enzyme is selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, and the like. orthologues of Cas9 from genera belonging to the group consisting of: Bacillus subtilis, ... The Cas comprises a helical domain 2 truncation.

157. 157. The composition of claim 156, wherein the CRISPR enzyme is truncated compared to a wild-type CRISPR enzyme, or wherein the CRISPR enzyme comprises between 500 and 2000 amino acids.

158. The composition of claim 156, wherein the CRISPR enzyme is a nuclease that directs cleavage of both strands at the location of the target sequence, or the CRISPR enzyme is a nickase that directs cleavage of one strand at the location of the target sequence.

159. 157. The composition of claim 156, wherein the guide sequence comprises at least 15 nucleotides.

160. The composition of claim 156, wherein the CRISPR enzyme is codon-optimized or is codon-optimized for expression in a eukaryotic cell.

161. The composition of claim 156, wherein the CRISPR enzyme comprises one or more mutations.

162. The composition of claim 156, wherein the CRISPR enzyme comprises a chimeric CRISPR enzyme.

163. 1. A method of modifying an organism or non-human organism by manipulation of a target sequence in a genomic locus of interest, comprising delivering a non-naturally occurring or engineered composition, said composition comprising: A)-I. A CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) a tracr mate sequence, and (c) tracr sequence A polynucleotide sequence comprising: II. Polynucleotide sequence encoding a CRISPR enzyme comprising at least one nuclear localization sequence Including, (a), (b) and (c) are arranged in a 5' to 3' orientation; the tracr mate sequence, when transcribed, hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence; the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence, and the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA; or (B) I. (a) a guide sequence capable of hybridizing to a target sequence in a prokaryotic cell, and (b) at least one tracr mate sequence; A polynucleotide comprising II. A polynucleotide sequence encoding a CRISPR enzyme, and III. Polynucleotide sequences containing tracr sequences Including, the tracr mate sequence, when transcribed, hybridizes to the tracr sequence, and the guide sequence directs sequence-specific target binding of a CRISPR complex to the target sequence; the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence hybridized to the target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence, and the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA; The CRISPR enzyme is selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, and the like. orthologues of Cas9 from genera belonging to the group consisting of: Bacillus subtilis, ... The method, wherein the Cas comprises a helical domain 2 truncation.

164. 164. The method of Claim 163, wherein the CRISPR enzyme is truncated compared to a wild-type CRISPR enzyme, or wherein the CRISPR enzyme comprises between 500 and 2000 amino acids.

165. 164. The method of claim 163, wherein the CRISPR enzyme is a nuclease that directs cleavage of both strands at the location of the target sequence, or wherein the CRISPR enzyme is a nickase that directs cleavage of one strand at the location of the target sequence.

166. 164. The method of Claim 163, wherein the guide sequence comprises at least 15 nucleotides.

167. 164. The method of claim 163, wherein the CRISPR enzyme is codon-optimized or is codon-optimized for expression in a eukaryotic cell.

168. 164. The method of Claim 163, wherein the CRISPR enzyme comprises one or more mutations.

169. 164. The method of claim 163, wherein the CRISPR enzyme comprises a chimeric CRISPR enzyme.

170. 1. A method of altering expression of one or more gene products, comprising introducing into a cell containing and expressing a DNA molecule encoding said one or more gene products an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein and one or more guide RNAs that target a DNA molecule, whereby said one or more guide RNAs target a genomic locus of said DNA molecule encoding said one or more gene products and said Cas protein cleaves the genomic locus of said DNA molecule encoding said one or more gene products, thereby altering expression of said one or more gene products; wherein said Cas protein and said guide RNA do not occur together in nature.

171. 1. A method for altering expression of one or more gene products, comprising introducing into a cell that contains and expresses a DNA molecule encoding said one or more gene products, an engineered non-naturally occurring vector system comprising one or more vectors, said vectors comprising: a) a first regulatory element operably linked to one or more CRISPR-Cas system guide RNAs that hybridize to a target sequence in a genomic locus of said DNA molecule encoding said one or more gene products; b) a second regulatory element operably linked to the Cas protein. Including, components (a) and (b) are located on the same or different vectors of said system, whereby the guide RNA targets the genomic locus of the DNA molecule encoding the one or more gene products and the Cas protein cleaves the genomic locus of the DNA molecule encoding the one or more gene products, thereby altering expression of the one or more gene products; the Cas protein and the guide RNA do not occur together in nature, The method, wherein the Cas comprises a helical domain 2 truncation.

172. 172. The method of claim 170 or 171, wherein the expression of more than one gene product is altered.

173. 173. The method of any one of claims 170 to 172, wherein the Cas protein of the vector or system further comprises one or more NLS.

174. 174. The method of any one of claims 170-173, wherein the guide RNA comprises a guide sequence fused to a tracr sequence.

175. 175. The method of any one of claims 170 to 174, wherein the cell is a eukaryotic cell.

176. 176. The method of claim 175, wherein the eukaryotic cell is a mammalian cell.

177. 177. The method of claim 176, wherein the mammalian cell is a human cell.

178. 178. The method of any one of claims 170-177, wherein the Cas protein is codon-optimized for expression in a eukaryotic cell.

179. 179. The method of any one of claims 170 to 178, wherein the expression of the gene product is reduced.

180. 180. The method of any one of claims 170 to 179, wherein the gene product is a protein.

181. 181. The method of any one of claims 170 to 180, wherein said introducing into said cell is by a delivery system comprising a viral particle, a liposome, electroporation, microinjection or conjugation.

182. 1. An engineered non-naturally occurring vector system comprising one or more vectors, The vector comprises: a) a first regulatory element operably linked to one or more CRISPR-Cas system guide RNAs that hybridize to a target sequence in a genomic locus of a DNA molecule encoding one or more gene products; b) a second regulatory element operably linked to the Cas protein. Including, components (a) and (b) are located on the same or different vectors of said system, whereby the guide RNA targets the genomic locus of the DNA molecule encoding the one or more gene products in the cell and the Cas protein cleaves the genomic locus of the DNA molecule encoding the one or more gene products, thereby altering expression of the one or more gene products; the Cas protein and the guide RNA do not occur together in nature, The Cas is a vector system that contains a helical domain 2 truncation.

183. 1. An engineered, programmable, non-naturally occurring CRISPR-Cas system comprising a Cas protein and one or more guide RNAs that target a genomic locus of a DNA molecule encoding one or more gene products in a cell, wherein the Cas protein cleaves the genomic locus of the DNA molecule encoding the one or more gene products, thereby altering expression of the one or more gene products; wherein the Cas protein and the guide RNA do not occur together in nature, and wherein the Cas comprises a helical domain 2 truncation.

184. 184. The system of claim 182 or 183, which alters expression of two or more gene products.

185. 184. The system of any one of claims 182 to 183, further comprising one or more NLS.

186. The system of any one of claims 182-185, wherein the guide RNA comprises a guide sequence fused to a tracr sequence.

187. The system of any one of claims 182 to 186, wherein the cell is a eukaryotic cell.

188. 188. The system of claim 187, wherein the eukaryotic cell is a mammalian cell.

189. The system of claim 188, wherein the mammalian cell is a human cell.

190. 190. The system of any one of claims 182 to 189, wherein the CRISPR-Cas system is a type II CRISPR-Cas system.

191. 191. The system of any one of claims 182 to 190, wherein the Cas protein is a Cas9 protein.

192. 192. The system of any one of claims 182 to 191, wherein the Cas protein is codon-optimized for expression in eukaryotic cells.

193. 193. The system of any one of claims 182 to 192, which reduces expression of said gene product.

194. The system according to any one of claims 182 to 193, wherein the gene product is a protein.

195. The system according to any one of claims 182 to 194, which is introduced into the cell by a delivery system comprising a viral particle, a liposome, electroporation, microinjection or conjugation.

196. A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors, The vector comprises: I. A first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, said polynucleotide sequence comprising: (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) a tracr mate sequence, and (c) tracr sequence A first regulatory element comprising: II. A second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme that includes at least one nuclear localization sequence (NLS) proximal to the end of the CRISPR enzyme. Including, (a), (b) and (c) are arranged in a 5' to 3' orientation; Components I and II are located on the same or different vectors of the system, the tracr mate sequence, when transcribed, hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence; The CRISPR complex comprises: (1) the guide sequence hybridized to the target sequence; and (2) the CRISPR enzyme complexed with the tracr mate sequence hybridized to the tracr sequence; the CRISPR enzyme comprises one or more mutations in a catalytic domain, thereby rendering the CRISPR enzyme a nickase that cleaves single-stranded DNA strands; said chimeric RNA polynucleotide sequence comprising two or more hairpins; The Cas comprises a helical domain 2 truncation.

197. The composition of claim 196, wherein multiple chiRNA polynucleotide sequences are used to provide a multiplexing system.

198. A multiplexed CRISPR enzyme system, comprising a vector system comprising one or more vectors, the vectors comprising: I. A first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, said polynucleotide sequence comprising: (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) a tracr mate sequence, and (c) tracr sequence A first regulatory element comprising: II. A second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme that includes at least one nuclear localization sequence (NLS) proximal to the end of the CRISPR enzyme. Including, (a), (b) and (c) are arranged in a 5' to 3' orientation; Components I and II are located on the same or different vectors of the system, the tracr mate sequence, when transcribed, hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence; The CRISPR complex comprises: (1) the guide sequence hybridized to the target sequence; and (2) the CRISPR enzyme complexed with the tracr mate sequence hybridized to the tracr sequence; the CRISPR enzyme comprises one or more mutations in a catalytic domain, thereby rendering the CRISPR enzyme a nickase that cleaves single-stranded DNA strands; the chiRNA polynucleotide sequence comprises two or more hairpins; In said multiplexing system, a plurality of chiRNA polynucleotide sequences are used, The Cas system comprises helical domain 2 truncation.

199. 200. The composition of claim 196, 197, or 198, wherein the first regulatory element is a polymerase III promoter.

200. 200. The composition or system of any one of claims 196 to 199, wherein the second regulatory element is a polymerase II promoter.

201. 201. The composition or system of any one of Claims 196-200, wherein the CRISPR enzyme comprises one or more NLS of sufficient strength to drive accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.

202. 202. The composition or system of any one of claims 196-201, wherein the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

203. 203. The composition or system of any one of claims 196 to 202, wherein the CRISPR enzyme is a Type II CRISPR system enzyme.

204. 204. The composition or system of any one of claims 196 to 203, wherein the CRISPR enzyme is a Cas9 enzyme.

205. 205. The composition or system of any one of claims 196-204, wherein the CRISPR enzyme is codon optimized for expression in a eukaryotic cell.

206. 206. The composition or system of any one of claims 196-205, wherein the guide sequence is at least 15 nucleotides in length.

207. 207. The composition or system of any one of claims 196-206, wherein said chimeric RNA polynucleotide sequence comprises 2, 3, 4 or 5 hairpins.

208. 208. The composition or system of any one of claims 196 to 207, wherein the catalytic domain is selected from the group comprising RuvCI, RuvCII, RuvCIII or HNH domains.

209. 209. The composition or system of any one of Claims 196-208, wherein the CRISPR enzyme comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986 of SpCas9, or corresponding residues in other CRISPR enzymes, or N580 with a SaCas9 protein.

210. A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors, said vectors comprising: I. (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) tracr mate sequence A first regulatory element operably linked to II. A second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme comprising at least one nuclear localization sequence (NLS) proximal to a terminus of the CRISPR enzyme; and III. A third regulatory element operably linked to the tracr sequence Including, Components I, II and III are located on the same or different vectors of said system, the tracr mate sequence, when transcribed, hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence; The CRISPR complex comprises: (1) the guide sequence hybridized to the target sequence; and (2) the CRISPR enzyme complexed with the tracr mate sequence hybridized to the tracr sequence; the CRISPR enzyme comprises one or more mutations in a catalytic domain, thereby rendering the CRISPR enzyme a nickase that cleaves single-stranded DNA strands; The Cas comprises a helical domain 2 truncation.

211. The composition of claim 210, wherein multiple guide sequences and a single tracr sequence are used to provide a multiplexing system.

212. A multiplexed CRISPR enzyme system, comprising a vector system comprising one or more vectors, the vectors comprising: I. (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) tracr mate sequence A first regulatory element operably linked to II. A second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme comprising at least one nuclear localization sequence (NLS) proximal to a terminus of the CRISPR enzyme; and III. A third regulatory element operably linked to the tracr sequence Including, Components I, II and III are located on the same or different vectors of said system, the tracr mate sequence, when transcribed, hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence; The CRISPR complex comprises: (1) the guide sequence hybridized to the target sequence; and (2) the CRISPR enzyme complexed with the tracr mate sequence hybridized to the tracr sequence; the CRISPR enzyme comprises one or more mutations in a catalytic domain, thereby rendering the CRISPR enzyme a nickase that cleaves single-stranded DNA strands; In the multiplexing system, multiple guide sequences and a single tracr sequence are used, The Cas system comprises helical domain 2 truncation.

213. 213. The composition or system of any one of claims 210 to 212, wherein the first regulatory element is a polymerase III promoter.

214. 214. The composition or system of any one of claims 210 to 213, wherein the second regulatory element is a polymerase II promoter.

215. 215. The composition or system of any one of claims 210 to 214, wherein the third regulatory element is a polymerase III promoter.

216. 216. The composition or system of any one of Claims 210-215, wherein the CRISPR enzyme comprises one or more NLS of sufficient strength to drive accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.

217. 163. The composition or system of any one of claims 156-162, wherein the tracr sequences exhibit at least 50% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

218. 218. The composition or system of any one of claims 210 to 217, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

219. 219. The composition or system of any one of claims 210 to 218, wherein the CRISPR enzyme is a Cas9 enzyme.

220. 220. The composition or system of any one of claims 210 to 219, wherein the CRISPR enzyme is codon optimized for expression in a eukaryotic cell.

221. 221. The composition or system of any one of claims 210-220, wherein the guide sequence is at least 15 nucleotides in length.

222. 222. The composition or system of claims 210-221, wherein the catalytic domain is selected from the group comprising RuvCI, RuvCII, RuvCIII or HNH domain.

223. 223. The composition or system of any one of Claims 210-222, wherein the CRISPR enzyme comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986 of SpCas9, or corresponding residues in other CRISPR enzymes or N580 with SaCas9 protein.

224. 224. A eukaryotic host cell comprising the composition of any one of claims 1-223.

225. 225. An organism comprising the eukaryotic host cell of claim 224.

226. A non-human organism comprising the eukaryotic host cell of claim 224.

227. A kit comprising the composition or system of any one of claims 1 to 226 and instructions for use of the kit.

228. 1. A method for altering expression of a genomic locus of interest in a eukaryotic cell, comprising: Contacting said genomic locus with a composition or system according to any one of claims 141 to 170; determining whether expression of said genomic locus is altered; Including, The method, wherein the Cas comprises a helical domain 2 truncation.

229. 1. A method for altering expression of one or more gene products, comprising: introducing into a eukaryotic cell that contains and expresses a DNA molecule encoding said one or more gene products, said vector comprising an engineered non-naturally occurring vector system comprising one or more vectors, said vector comprising: a) a first regulatory element operably linked to one or more clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system guide RNAs that hybridize to a target sequence in a genomic locus of said DNA molecule encoding said one or more gene products; b) a second regulatory element operably linked to the type II Cas9 protein. Including, components (a) and (b) are located on the same or different vectors of said system, whereby the guide RNA targets the genomic locus of the DNA molecule encoding the one or more gene products and the Cas9 protein cleaves the genomic locus of the DNA molecule encoding the one or more gene products, thereby altering expression of the one or more gene products; the Cas9 protein and the guide RNA do not occur together in nature, The method, wherein the Cas comprises a helical domain 2 truncation.

230. 230. The method of claim 229, wherein the expression of two or more gene products is altered.

231. 231. The method of claim 229 or 230, wherein the vector of the system further comprises one or more nuclear localization signals (NLS).

232. 232. The method of any one of Claims 229-231, wherein the guide RNA comprises a guide sequence fused to a tracr sequence.

233. 233. The method of any one of claims 229-232, wherein the Cas9 protein is codon-optimized for expression in a eukaryotic cell.

234. The method of any one of claims 229 to 233, wherein the eukaryotic cell is a mammalian or human cell.

235. 230. The method of claim 229, wherein the expression of one or more gene products is reduced.

236. 1. An engineered non-naturally occurring vector system comprising one or more vectors, said vectors comprising: a) a first regulatory element operably linked to one or more CRISPR-Cas system guide RNAs that hybridize to a target sequence in a genomic locus of a DNA molecule encoding one or more gene products; b) a second regulatory element operably linked to the type II Cas9 protein. Including, components (a) and (b) are located on the same or different vectors of said system, whereby the guide RNA targets the genomic locus of the DNA molecule encoding the one or more gene products in a eukaryotic cell and the Cas9 protein cleaves the genomic locus of the DNA molecule encoding the one or more gene products, thereby altering expression of the one or more gene products; the Cas9 protein and the guide RNA do not occur together in nature, The Cas system comprises helical domain 2 truncation.

237. 237. The vector system of claim 236, which alters expression of two or more gene products.

238. 238. A vector system according to claim 236 or 237, wherein the vector of the system further comprises one or more NLS.

239. 239. The vector system of claims 236, 237, or 238, wherein the guide RNA comprises a guide sequence fused to a tracr sequence.

240. 240. The vector system of claims 236, 237, 238 or 239, wherein the Cas9 protein is codon-optimized for expression in the eukaryotic cell.

241. 241. The vector system of claim 236, 237, 238, 239 or 240, wherein the eukaryotic cell is a mammalian or human cell.

242. 242. The vector system of any one of claims 236 to 241, which reduces expression of one or more gene products.

243. 1. An engineering programmable non-naturally occurring Type II CRISPR-Cas system comprising a Cas9 protein and one or more guide RNAs that target a genomic locus of a DNA molecule encoding one or more gene products in a eukaryotic cell, wherein the Cas9 protein cleaves the genomic locus of the DNA molecule encoding the one or more gene products, thereby altering expression of the one or more gene products; wherein the Cas9 protein and the guide RNA do not occur together in nature, and wherein the Cas comprises a Helical Domain 2 truncation.

244. 244. The CRISPR-Cas system of claim 243, wherein expression of two or more gene products is altered.

245. The CRISPR-Cas system of claim 243 or 244, further comprising one or more NLS.

246. 246. The CRISPR-Cas system of Claims 243, 244, or 245, wherein the guide RNA comprises a guide sequence fused to a tracr sequence.

247. 246. The CRISPR-Cas system of claims 243, 244 or 245, wherein the Cas9 protein is codon-optimized for expression in a eukaryotic cell.

248. The CRISPR-Cas system of any one of claims 243-247, wherein the eukaryotic cell is a mammalian or human cell.

249. 249. The composition, method or use of any one of claims 56-117, 125-169, 171-248, wherein the helical domain 2 truncation is replaced by one or more sets of flexible glycine-serine (GlyGlyGlySer) or rigid alpha-helical linkers (Ala(GluAlaAlaAlaLys)Ala) of groups of 3, 6, 9, or 12 repeats to replace helical domain 2 for potential structural stabilization and / or to assist in retaining Cas9:sgRNA specificity.

250. The composition, method or use of any one of claims 2 to 248, wherein the Cas is SpCas.

251. The composition, method or use of claim 207, wherein the SpCas is SpCas9.

252. A non-naturally occurring CRISPR enzyme, wherein the HD2 domain is truncated.

253. The CRISPR enzyme of claim 252, comprising Cas9.

254. The CRISPR enzyme of claim 253, wherein the Cas9 comprises SpCas9.

255. The CRISPR enzyme of claim 253, wherein the Cas9 comprises SaCas9.

256. 256. The CRISPR enzyme of any one of claims 252-255, wherein the truncation is a substitution of the HD2 domain.

257. 257. The CRISPR enzyme of claim 256, wherein the truncation is a replacement of the HD2 domain with a linker.

258. The CRISPR enzyme of claim 257, wherein the linker is a flexible linker.

259. The CRISPR enzyme of claim 258, wherein the flexible linker comprises a GlySer linker.

260. 260. The composition, method, system or use of any one of claims 2 to 248, wherein the CRISPR enzyme is as defined in any one of claims 252 to 259.

261. A chimeric three component CRISPR enzyme comprising N'- and C'-terminal components from a first CRISPR enzyme and an internal component from a second CRISPR enzyme, said second CRISPR enzyme being an orthologue of said first CRISPR enzyme.

262. 262. The CRISPR enzyme of claim 261, wherein the first and second CRISPR enzymes each comprise Cas9.

263. 263. The CRISPR enzyme of claim 262, wherein the first and second Cas9 comprise SpCas9.

264. The CRISPR enzyme of claim 262, wherein the first and second Cas9 comprise SaCas9.

265. The CRISPR enzyme of claim 262, wherein the first and second Cas9 comprise StCas9.

266. The CRISPR enzyme of claim 265, wherein the StCas9 comprises St3Cas9.

267. 267. The CRISPR enzyme of any one of claims 261-266, wherein an internal component from the first CRISPR enzyme is replaced by an internal component from the second CRISPR enzyme, said internal components being the same or different.

268. The CRISPR enzyme of any one of claims 261 to 267, wherein the second CRISPR enzyme is a SaCRISPR enzyme.

269. 268. The CRISPR enzyme of any one of claims 261-267, wherein the second CRISPR enzyme is a StCRISPR enzyme.

270. The CRISPR enzyme of claim 269, wherein the StCRISPR enzyme is a St3CRISPR enzyme.

271. 270. The composition, method, system or use of any one of claims 2 to 248, wherein the CRISPR enzyme is as defined in any one of claims 261 to 270.