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Engineering and optimization of systems, methods and compositions for sequence … — The Broad Institute, Inc. (US20250250577A1)

ABSTRACT

Abstract

The invention provides for engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors with additional functional domains. Also provided are methods of directing CRISPIR complex formation in prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.

Description

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. patent application Ser. No. 19/025,722, filed Jan. 16, 2025, which is a continuation of U.S. patent application Ser. No. 14/738,398, filed Jun. 12, 2015, which is a continuation-in-part of international patent application serial no. PCT/US2013/074736 filed Dec. 12, 2013 and published as WO 2014/093655, which claims priority to U.S. provisional patent application 61/835,936 entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH FUNCTIONAL DOMAINS FILED ON Jun. 17, 2013, and which also claims priority to U.S. provisional patent applications 61/758,468; 61/769,046; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130 each entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Jan. 30, 2013; Feb. 25, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013 respectively, and which also claims priority to U.S. provisional patent applications 61/736,527 and 61/748,427, both entitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec. 12, 2012 and Jan. 2, 2013, respectively, and which also claims priority to U.S. provisional patent applications 61/791,409 and 61/835,931 both entitled BI-2011/008/44790.02.2003 and BI-2011/008/44790.03.2003 filed on Mar. 15, 2013 and Jun. 17, 2013 respectively.

Reference is also made to international patent applications PCT/US2013/074611 filed Dec. 12, 2013 and published as WO 2014/093595; PCT/US2013/074743 filed Dec. 12, 2013 and published as WO 2014/093661; PCT/US2013/074790 filed Dec. 12, 2013 and published as WO 2014/093694; PCT/US2013/074825 filed Dec. 12, 2013 and published as WO 2014/093718; PCT/US2013/074812 filed Dec. 12, 2013 and published as WO 2014/093709; PCT/US2013/074667 filed Dec. 12, 2013 and published as WO 2014/093622; PCT/US2013/074691 filed Dec. 12, 2013 and published as WO 2014/093635; PCT/US2013/074819 filed Dec. 12, 2013 and published as WO 2014/093712; and PCT/US2013/074800 filed Dec. 12, 2013 and published as WO 2014/093701.

Reference is also made to U.S. provisional patent applications 61/757,972 filed Jan. 29, 2013, 61/799,800 filed Mar. 15, 2013; 61/835,936, 61/835,973, 61/836,080, 61/836,101, 61/836,123, 61/836,127, and 61/847,537, each filed Jun. 17, 2013; 61/862,468 and 61/862,355, each filed Aug. 5, 2013; 61/871,301 filed Aug. 28, 2013; 61/969,777 filed Sep. 25, 2013; and 61/961,980 filed Oct. 28, 2013.

The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under the NIH Pioneer Award (1DP1MH100706) awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

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

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Apr. 16, 2025, is named 114203-6219_SL.xml and is 4,728 bytes in size.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome.

SUMMARY OF THE INVENTION

The CRISPR/Cas or the CRISPR-Cas system (both terms are used interchangeably throughout this application) does not require the generation of customized proteins to target specific sequences but rather a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target, in other words the Cas enzyme can be recruited to a specific DNA target using said short RNA molecule. Adding the CRISPR-Cas system to the repertoire of genome sequencing techniques and analysis methods may significantly simplify the methodology and accelerate the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. To utilize the CRISPR-Cas system effectively for genome editing without deleterious effects, it is critical to understand aspects of engineering and optimization of these genome engineering tools, which are aspects of the claimed invention.

There exists a pressing need for alternative and robust systems and techniques for sequence targeting with a wide array of applications. Aspects of this invention address this need and provide related advantages. An exemplary CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.

In one aspect, the invention relates to one or more elements of a CRISPR system having improved or modified functionality. The CRISPR complex of the invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the invention has a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types. As such the CRISPR complex of the invention has a broad spectrum of applications in, e.g., gene or genome editing, gene therapy, drug discovery, drug screening, disease diagnosis, and prognosis. An exemplary CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.

In some embodiments, a CRISPR/Cas system may comprise: (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex may comprise a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme which may comprise a nuclear localization sequence; wherein components (a) and (b) are located on the same or different vectors of the system. In some embodiments, component (a) further may comprise the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further may comprise two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system may comprise the tracr sequence under the control of a third regulatory element, such as a polymerase III promoter. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, the CRISPR enzyme may comprise one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. Without wishing to be bound by theory, it is believed that a nuclear localization sequence is not necessary for CRISPR activity in eukaryotes, but that including such sequences enhances activity of the system. 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. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from either of these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length. In general, and throughout this specification, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector may comprise one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the 3-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Aspects of the invention relate to methods of improving or modifying the target specificity of a CRISPR enzyme (preferably a Cas9 enzyme) which may comprise: a) selecting the CRISPR enzyme having a smaller size for easy packaging into delivery vectors; or b) generating chimeric CRISPR enzymes; or c) utilizing mutated CRISPR (preferably Cas9) enzymes. Further aspects of the invention also relate to methods and compositions for improving target specificity involving two CRISPR enzymes (double nickase), improving the sgRNA scaffold by making improvement to other components aside from the guide RNA.

The invention comprehends a non-naturally occurring or engineered composition comprising a vector system that may comprise one or more vectors comprising I. a first regulatory element operably linked to a CRISPR/Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence may comprise (a) a guide sequence capable of hybridizing to a target sequence in a cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences, wherein (a), (b) and (c) are arranged

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. patent application Ser. No. 19/025,722, filed Jan. 16, 2025, which is a continuation of U.S. patent application Ser. No. 14/738,398, filed Jun. 12, 2015, which is a continuation-in-part of international patent application serial no. PCT/US2013/074736 filed Dec. 12, 2013 and published as WO 2014/093655, which claims priority to U.S. provisional patent application 61/835,936 entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH FUNCTIONAL DOMAINS FILED ON Jun. 17, 2013, and which also claims priority to U.S. provisional patent applications 61/758,468; 61/769,046; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130 each entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Jan. 30, 2013; Feb. 25, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013 respectively, and which also claims priority to U.S. provisional patent applications 61/736,527 and 61/748,427, both entitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec. 12, 2012 and Jan. 2, 2013, respectively, and which also claims priority to U.S. provisional patent applications 61/791,409 and 61/835,931 both entitled BI-2011/008/44790.02.2003 and BI-2011/008/44790.03.2003 filed on Mar. 15, 2013 and Jun. 17, 2013 respectively.

Reference is also made to international patent applications PCT/US2013/074611 filed Dec. 12, 2013 and published as WO 2014/093595; PCT/US2013/074743 filed Dec. 12, 2013 and published as WO 2014/093661; PCT/US2013/074790 filed Dec. 12, 2013 and published as WO 2014/093694; PCT/US2013/074825 filed Dec. 12, 2013 and published as WO 2014/093718; PCT/US2013/074812 filed Dec. 12, 2013 and published as WO 2014/093709; PCT/US2013/074667 filed Dec. 12, 2013 and published as WO 2014/093622; PCT/US2013/074691 filed Dec. 12, 2013 and published as WO 2014/093635; PCT/US2013/074819 filed Dec. 12, 2013 and published as WO 2014/093712; and PCT/US2013/074800 filed Dec. 12, 2013 and published as WO 2014/093701.

Reference is also made to U.S. provisional patent applications 61/757,972 filed Jan. 29, 2013, 61/799,800 filed Mar. 15, 2013; 61/835,936, 61/835,973, 61/836,080, 61/836,101, 61/836,123, 61/836,127, and 61/847,537, each filed Jun. 17, 2013; 61/862,468 and 61/862,355, each filed Aug. 5, 2013; 61/871,301 filed Aug. 28, 2013; 61/969,777 filed Sep. 25, 2013; and 61/961,980 filed Oct. 28, 2013.

The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under the NIH Pioneer Award (1DP1MH100706) awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

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

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Apr. 16, 2025, is named 114203-6219_SL.xml and is 4,728 bytes in size.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome.

SUMMARY OF THE INVENTION

The CRISPR/Cas or the CRISPR-Cas system (both terms are used interchangeably throughout this application) does not require the generation of customized proteins to target specific sequences but rather a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target, in other words the Cas enzyme can be recruited to a specific DNA target using said short RNA molecule. Adding the CRISPR-Cas system to the repertoire of genome sequencing techniques and analysis methods may significantly simplify the methodology and accelerate the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. To utilize the CRISPR-Cas system effectively for genome editing without deleterious effects, it is critical to understand aspects of engineering and optimization of these genome engineering tools, which are aspects of the claimed invention.

There exists a pressing need for alternative and robust systems and techniques for sequence targeting with a wide array of applications. Aspects of this invention address this need and provide related advantages. An exemplary CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.

In one aspect, the invention relates to one or more elements of a CRISPR system having improved or modified functionality. The CRISPR complex of the invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the invention has a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types. As such the CRISPR complex of the invention has a broad spectrum of applications in, e.g., gene or genome editing, gene therapy, drug discovery, drug screening, disease diagnosis, and prognosis. An exemplary CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.

In some embodiments, a CRISPR/Cas system may comprise: (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex may comprise a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme which may comprise a nuclear localization sequence; wherein components (a) and (b) are located on the same or different vectors of the system. In some embodiments, component (a) further may comprise the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further may comprise two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system may comprise the tracr sequence under the control of a third regulatory element, such as a polymerase III promoter. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, the CRISPR enzyme may comprise one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. Without wishing to be bound by theory, it is believed that a nuclear localization sequence is not necessary for CRISPR activity in eukaryotes, but that including such sequences enhances activity of the system. 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. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from either of these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length. In general, and throughout this specification, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector may comprise one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the 3-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Aspects of the invention relate to methods of improving or modifying the target specificity of a CRISPR enzyme (preferably a Cas9 enzyme) which may comprise: a) selecting the CRISPR enzyme having a smaller size for easy packaging into delivery vectors; or b) generating chimeric CRISPR enzymes; or c) utilizing mutated CRISPR (preferably Cas9) enzymes. Further aspects of the invention also relate to methods and compositions for improving target specificity involving two CRISPR enzymes (double nickase), improving the sgRNA scaffold by making improvement to other components aside from the guide RNA.

The invention comprehends a non-naturally occurring or engineered composition comprising a vector system that may comprise one or more vectors comprising I. a first regulatory element operably linked to a CRISPR/Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence may comprise (a) a guide sequence capable of hybridizing to a target sequence in a cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences, wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation, wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, wherein the CRISPR enzyme may comprise two or more mutations, such that the enzyme has altered nuclease activity compared with the wild type enzyme, and wherein the enzyme-coding sequence further encodes one or more heterologous functional domains.

The invention further comprehends a multiplexed two component CRISPR enzyme system composition comprising a vector system that may comprise one or more vectors comprising I. a first regulatory element operably linked to a CRISPR/Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to a target sequence in a cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme may comprise at least one or more nuclear localization sequences, wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation, wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, wherein the CRISPR enzyme comprises two or more mutations, such that the enzyme has altered nuclease activity compared with the wild type enzyme, wherein the enzyme-coding sequence further encodes one or more heterologous functional domains, and wherein in the multiplexed system composition multiple chiRNA polynucleotide sequences are used.

The invention further comprehends a non-naturally occurring or engineered composition comprising a vector system that may comprise one or more vectors comprising I. a first regulatory element operably linked to (a) a guide sequence capable of hybridizing to a target sequence in a cell, and (b) at least one or more tracr mate sequences, II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and III. a third regulatory element operably linked to a tracr sequence, wherein components I, II and III are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, wherein the CRISPR enzyme may comprise two or more mutations, such that the enzyme has altered nuclease activity compared with the wild type enzyme, and wherein the enzyme-coding sequence further encodes one or more heterologous functional domains.

The invention also comprehends a multiplexed three component CRISPR enzyme system composition comprising a vector system that may comprise one or more vectors comprising I. a first regulatory element operably linked to (a) a guide sequence capable of hybridizing to a target sequence in a cell, and (b) at least one or more tracr mate sequences, II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and III. a third regulatory element operably linked to a tracr sequence, wherein components I, II and III are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, wherein the CRISPR enzyme comprises two or more mutations, such that the enzyme has altered nuclease activity compared with the wild type enzyme, wherein the enzyme-coding sequence further encodes one or more heterologous functional domains, and wherein in the multiplexed system composition multiple guide sequences capable of hybridizing to multiple target sequences are used.

In embodiments of the invention the CRISPR enzyme may comprise one or more mutations in two or more catalytically active domains. In another embodiment the CRISPR enzyme has reduced or abolished nuclease activity compared with the wild type enzyme. In another embodiment the two mutations are D10A SpCas9 in a first catalytically active domain and H840A SpCas9 in a second catalytically active domain or corresponding residues of other CRISPR enzymes. In another embodiment the CRISPR enzyme may comprise two or more mutations in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986. In another embodiment the CRISPR enzyme may comprise two or more mutations selected from the group comprising D10A, E762A, H840A, N854A, N863A or D986A. In a preferred embodiment the CRISPR enzyme is a DNA binding protein that does not direct cleavage of either strand at the location of the target sequence. In an embodiment, each of the two or more mutations is in a catalytically active domain of the CRISPR enzyme selected from the group comprising RuvCI, RuvCII, RuvCIII or HNH domain. It will be understood throughout this application that many references to specific amino acid residues are to those of the SpCas9 enzyme. The skilled person will understand that the invention is applicable to other Cas enzymes, including Cas9 enzymes from other sources, and that where reference is made to specific SpCas9 enzyme residues corresponding alterations and mutations may be made in those other Cas enzymes. For example, the skilled person will be able to compare sequences of the SpCas9 and other enzymes, and identify corresponding residues and domains, and hence to make appropriate modifications to those enzymes.

In further embodiments, the compositions of the invention may comprise at least two or more nuclear localization sequences. In an aspect of the invention the functional domain is a transcriptional activation domain, e. g. VP64. In another aspect the functional domain is a transcriptional repressor domain, e.g. a KRAB domain, a SID domain or a SID4X domain. In another embodiment, the enzyme coding sequence encodes one, two, three, four, five or more heterologous functional domains fused to the CRISPR enzyme. The invention also comprehends one or more linker sequences between any two domains. Embodiments of the invention include one or more functional domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. In another embodiment, the functional domain binds DNA and/or affects transcription of the target nucleic acid. In aspects of the invention the cell is a prokaryotic or eukaryotic cell. In a preferred embodiment, the cell is a mammalian cell or a human cell. In some embodiments, the mammalian cell may be a rodent (for example, mouse or rat) cell, an ungulate cell, or a primate cell. In some embodiments, the eukaryotic cell may be an arthropod (eg, insect) or nematode cell. In other embodiments the cell may be a plant cell (including algae) or a fungal cell. In further embodiments of the invention the CRISPR enzyme is codon optimized for expression in a eukaryotic cell; the CRISPR enzyme is a type II CRISPR enzyme; the CRISPR enzyme is a Cas9 enzyme and the Cas9 enzyme is from an organism selected from the group comprising of genus Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter , Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium or Corynebacter . In a further aspect, the vectors of the system are viral vectors selected from the group comprising of a lentiviral vector, an adenoviral vector or an AAV vector.

The invention also comprehends methods of modulating, i.e. altering, activating, repressing, gene expression at a genomic locus of interest in a cell by contacting the cell with compositions of the invention.

An aspect of the invention relates to a composition which may comprise a vector system which may comprise one or more vectors which may comprise

I. a first regulatory element operably linked to a CRISPR/Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence may comprise (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme which may comprise at least one or more nuclear localization sequences, wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation, wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex may comprise the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and wherein the enzyme coding sequence encoding the CRISPR enzyme further encodes a heterologous functional domain.

The coding sequence may encode one or more heterologous functional domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein which may comprise a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In Chalasani, US2011/0059502, it is stated, “In certain embodiments, the activity of a second domain in a fusion protein can be about 25% to about 90% more specific than the activity of a second domain not in a fusion protein. In some embodiments, a second domain can comprise an endonuclease activity. In certain embodiments, a second domain can comprise a type II endonuclease activity . . . type IIs endonuclease activity (e.g., Fok I . . . ).” Accordingly, a functional domain as herein can be a FokI domain. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.

In an aspect of the invention, the functional domain affects transcription of the target nucleic acid.

Non-limiting examples of CRISPR enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database (available at the website uniprot.org) under accession number Q99ZW2. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.

In some aspects of the invention, a CRISPR enzyme may comprise one or more mutations and may be used as a generic DNA binding protein with or without fusion to a functional domain. The mutations may include but are not limited to catalytic mutations, for instance mutations in one of the catalytic domains or mutation of catalytic residues. Preferred examples of suitable catalytic mutations are the catalytic residue(s) in the N-term RuvC I domain of Cas9 or the catalytic residue(s) in the internal HNH domain. In some embodiments, the Cas9 is (or is derived from) SpCas9. In such embodiments, preferred mutations are at any or all or positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 or corresponding positions in other Cas9s (which may be ascertained for instance by standard sequence comparison tools). In particular, any or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged. The same (or conservative substitutions of these mutations) at corresponding positions in other Cas9s are also preferred. Particularly preferred are D10 and H840 in SpCas9. However, in other Cas9s, residues corresponding to SpCas9 D10 and H840 are also preferred.

In a more advantageous aspect of the invention the mutated CRISPR enzyme may be fused to a transcriptional activation domain. In one aspect of the invention, the transcriptional activation domain may be VP64. Other aspects of the invention relate to the mutated CRISPR enzyme being fused to domains which include but are not limited to a transcriptional repressor, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, a cryptochrome, a light inducible/controllable domain or a chemically inducible/controllable domain.

In some aspects of the invention, the CRISPR enzyme is comprised of less than four thousand, and in some aspects less than one thousand, amino acids. Such enzymes may be provided fused to a heterologous functional domain, or not. Where the enzyme is fused to a heterologous functional domain, the size of the enzyme (less than four thousand, less than one thousand amino acids) refers to the CRISPR portion of the fusion protein. In certain embodiments described herein, the invention or method is practiced on an organism or subject. In certain embodiments, the organism or subject is a eukaryote or a non-human eukaryote. In certain embodiments, the organism or subject is a plant. In certain embodiments, the organism or subject is a mammal or a non-human mammal. In certain embodiments, the organism or subject is algae. In some embodiments, the organism or subject may be a rodent (for example, mouse or rat), an ungulate, or a primate. In some embodiments, the organism or subject may be an arthropod (eg, insect) or nematode. In other embodiments the organism or subject may be a plant or a fungus.

Accordingly, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 shows a schematic of RNA-guided Cas9 nuclease. The Cas9 nuclease from Streptococcus pyogenes (yellow) is targeted to genomic DNA by a synthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue) and a scaffold (red). The guide sequence base-pairs with the DNA target (blue), directly upstream of a requisite 5′-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB) ˜3 bp upstream of the PAM (red triangle).

FIGS. 2 A- 2 F show an exemplary CRISPR system, a possible mechanism of action, an example adaptation for expression in eukaryotic cells, and results of tests assessing nuclear localization and CRISPR activity

FIG. 3 A-D is a phylogenetic tree of Cas genes

FIGS. 4 A- 4 F show the phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (˜1400 amino acids) and two of small Cas9s (˜1100 amino acids).

FIG. 5 shows a schematic construct in which the transcriptional activation domain (VP64) is fused to Cas9 with two mutations in the catalytic domains (D10 and H840).

FIG. 6 shows a graphical representation of transcriptional activation following co-transfection of the Cas9-VP64 with PCR generated chimeric crispr RNA (chiRNA) in 293 cells. Assessment was carried out 72 hours after transfection using RT-qPCR.

FIG. 7 shows a number of vectors incorporating mutant Cas9 genes with VP64, NLS, and GFP markers.

FIG. 8 shows the localization of Cas9-VP64-GFP constructs into 293 cells as assessed by a fluorescent microscope 12 hours post transfection.

FIG. 9 shows the localization of 16 dCas9-GFP fusions with the same alpha importin NLS sequence on either the N- or C-term looking at zero to three tandem repeats. Each construct was transfected into HEK 293FT cells using Lipofectame 2000 and imaged 24 hours post-transfection

FIG. 10 shows six versions of a 6×His tag added to dCas9, transfected into 293FT cells, and stained with an anti-6×His antibody. Three were constructed for transcriptional activation (VP64 fusions), and the other three were for transcriptional repression (no functional domain).

FIG. 11 shows a titrated ratio of chiRNA (Sox2.1 and Sox2.5) to Cas9 (NLS-VP64-NLS-hSpCas9-NLS-VP64-NLS), transfected into 293 cells, and quantified using RT-qPCR.

FIG. 12 shows the positioning of target sites in the human Sox2 locus with each target being 20 bp long with a neighboring NGG protspacer adjacent motif (PAM).

FIG. 13 shows co-transfecting of each dCas9 containing construct with pA6 plasmids into HEK 293FT cells using Lipofectame 2000. 72 hours post-transfection total RNA was extracted from the cells. 1 μg of RNA was reverse transcribed into cDNA (qScript Supermix) in a 40 ul reaction. 2 ul of reaction product was added into a single 20 ul TaqMan assay qPCR reaction. Each experiment was performed in biological and technical triplicates. No RT control and no template control reactions showed no amplification.

FIG. 14 shows testing of constructs (pXRP011, pXRP013, pXRP015) using the same Sox2 targets as FIG. 13 .

FIGS. 15 A- 15 B shows a list of 31 constructs to explore different linkers, functional domains, and N- and C-term fusions and critical elements.

FIG. 16 shows co-transfecting of each dCas9 repressor plasmid with two guide RNAs targeted to the coding strand of the beta-catenin gene. RNA was isolated 72 hours after transfection and gene expression was quantified by RT-qPCR. The endogenous control gene was GAPDH. Two validated shRNAs were used as positive controls. Negative controls were certain plasmids transfected without gRNA, these are denoted as “pXRP ##control”.

FIG. 17 shows a graphical representation of transcriptional activation further to the ratio of chiRNA (Sox2.1 and Sox2.5) to Cas9 (NLS-VP64-NLS-hSpCas9-NLS-VP64-NLS) being titrated and transfected in 293 cells. Results were quantified using Rt-qPCR.

FIG. 18 shows luciferase reporter data for Cas9 activator (top panel) and repressor (bottom panel). Compared to “No Cas9” controls over 3 fold activation was achieved when targeting the promoter of Sox2. When targeting the gene body of beta-catenin (CTNNB1), about 3 fold repression was achieved.

FIG. 19 shows gene expression of beta-catenin in HEK 293FT cells 72 hours after transfection. Cas9 repressor constructs were targeted to the beta-catenin locus and compared to the gold standard shRNAs. Similar repression could be seen with Cas9 repressors and the shRNA.

FIG. 20 shows a graphical representation of the fold Neurog2 expression of the 20 bp sgRNA sequences were targeted to the Neurog2 locus in mouse Neuro 2A cells. Neurog2 mRNA levels were measured using RT-qPCR.

FIG. 21 shows the basal gene expression modulation of dCas9-VP64 that was measured for the indicated gene targets. Italics represent mouse gene targets tested in Neuro 2A cells, all capsrepresent human gene targets tested in 293FT cells. In each case, expression of the gene in GFP transfected cells was compared to expression in cells transfected with Cas9-VP64.

FIG. 22 shows the changes in expression level of the indicated genes when samples transfected with the dCas9-VP64 construct, but without an sgRNA.

FIG. 23 A-G shows a, The RNA-guided nuclease Cas9 from the type II Streptococcus pyogenes CRISPR/Cas system can be converted into a nucleolytically inactive RNA-guided DNA binding protein (Cas9**) by introducing two alanine substitutions (D10A and H840A). Schematic showing that a synthetic guide RNA (sgRNA) can direct Cas9**-effector fusion to a specific locus in the human genome. The sgRNA contains a 20-bp guide sequence at the 5′ end which specifies the target sequence. On the target genomic DNA, the 20-bp target site needs to be followed by a 5′-NGG PAM motif b, c, Schematics showing the sgRNA target sites in the human KLF4 and SOX2 loci, respectively. Each target site is indicated by the blue bar and the corresponding PAM sequence is indicated by the magenta bar. d, e, Schematics of the Cas9**-VP64 transcription activator and SID4X-Cas9** transcription repressor constructs. f, g, Cas9**-VP64- and SID4X-Cas9**-mediated activation of KLF4 and repression of SOX2, respectively. All mRNA levels were measured relative to GFP mock-transfected 293FT cells (mean±s.e.m.; n=3 biological replicates).

FIG. 24 shows the Cas9 activator sequence.

FIG. 25 shows the Cas9 repressor sequence.

FIG. 26 shows the graphical representation of fold activation of different genes targeted by the Cas9 activator (pXRP57) and guide RNA.

FIG. 27 shows the graphical representation of fold repression of the hSox2 gene targeted by the Cas9 activator (pXRP57) and guide RNA.

FIG. 28 shows a pAAV-EF1a-dCas9-GS-CIB1(mNLS d318-334)_WPRE_hGHpolyA plasmid map.

FIG. 29 shows a pAAV-EF1a-dCas9-GS-NLS-cib1-WPRE-hGHpA plasmid map.

FIG. 30 shows a pAAV-EF1a-dCas9-GS-NLS-NLS-cib1-WPRE-hGHpA plasmid map.

FIG. 31 shows a graphical representation of CasLITE constructs exhibiting varying levels of light-inducible transcriptional activation.

FIG. 32 shows the validation of Rosa26 Cre-dependent Cas9 knockin mouse embryonic stem cells.

FIG. 33 shows a gel image indicating the genotyping results for Cas9 mice.

FIG. 34 shows CRISPR/Cmr mediated silencing of RNA. Cmr proteins form a complex with mature crRNA to site-specifically target and cleave RNA. Mature crRNAs from P. furiosus exist in two forms of different lengths, long (45 nt) and short (39 nt), consisting of a 5′ handle and a guide sequence. The 5′ handle is required for the crRNA to be included in the Cmr complex. The guide sequence programs the target site and can be either long (37 nt) or short (31 nt). RNA cleavage occurs 14 nucleotides from the 3′ end of the crRNA. This platform could be repurposed to target mammalian genes expressed from a genomic locus by only changing the guide sequence.

FIG. 35 A-B shows Cmr proteins are expressed within mammalian cells. Six Cmr genes (Cmr1-6) and one Cas gene (Cas6) from P. furiosus were cloned into mammalian expression vectors and transfected into HEK 293FT cells. 72 hours post-transfection fluorescence images were taken and protein lysates were prepared. A) EGFP expression is strongly observed suggesting that the exogenous proteins are robustly expressed. B) Western blot images show protein bands located at the expected size. The second band within each lane is 30 kDa larger in each case and is likely the result of uncleaved P2A sequence.

FIG. 36 shows CRISPR/Cmr expression vectors.

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

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the engineering and optimization of systems, methods and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to the CRISPR/Cas system and components thereof. In advantageous embodiments, the Cas enzyme is Cas9; for example, Cas9 from S. pyogenes or S. thermophilus.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The term also encompasses nucleic-acid-like structures with synthetic backbones, see, e.g., Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

In aspects of the invention the terms “chimeric RNA”, “chimeric guide RNA”, “guide RNA”, “single guide RNA” and “synthetic guide RNA” are used interchangeably and refer to the polynucleotide sequence comprising the guide sequence, the tracr sequence and the tracr mate sequence. The term “guide sequence” refers to the about 20 bp sequence within the guide RNA that specifies the target site and may be used interchangeably with the terms “guide” or “spacer”. The term “tracr mate sequence” may also be used interchangeably with the term “direct repeat(s)”.

As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. For example, “wild type StCas9” refers to wild type Cas9 from S thermophilus , the protein sequence of which is given in the SwissProt database under accession number G3ECR1. Similarly, S pyogenes Cas9 is included in SwissProt under accession number Q99ZW2.

As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature.

The terms “non-naturally occurring” or “engineered” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,

CLAIMS

Claims ( 20 )

1 . (canceled)

2 . A method for editing a eukaryotic cell, comprising delivering an engineered CRISPR-Cas system into the eukaryotic cell, wherein the engineered CRISPR-Cas system comprises:

(a) a Cas9 protein or a polynucleotide encoding the Cas9 protein, wherein the Cas9 protein is S. pyogenes Cas9 and is fused with at least one nuclear localization signal (NLS) and at least one heterologous protein domain; (b) a CRISPR-Cas system chimeric RNA or a polynucleotide encoding the chimeric RNA, wherein the chimeric RNA comprises a guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a genomic locus of interest of the eukaryotic cell, a tracr-mate sequence capable of hybridizing to a tracr sequence, and a tracr sequence comprising 40 or more nucleotides in length; wherein a CRISPR complex comprising the Cas9 protein and the chimeric RNA is formed in the eukaryotic cell, and wherein the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence adjacent to the PAM in the genomic locus of interest of the eukaryotic cell.

3 . The method of claim 2 , wherein the PAM is NGG.

4 . The method of claim 2 , wherein the tracr sequence comprises 50 or more nucleotides in length.

5 . The method of claim 2 , wherein the chimeric RNA further comprises a poly-U sequence.

6 . The method of claim 2 , wherein the chimeric RNA is encoded by SEQ ID NO:27.

7 . The method of claim 2 , wherein the chimeric RNA comprises one or more modified nucleotides.

8 . The method of claim 2 , wherein the chimeric RNA comprises one or more methylated nucleotides or nucleotide analogs.

9 . The method of claim 2 , wherein the NLS is independently selected from the group consisting of PKKKRKV, KRPAATKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSP, NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK

10 . The method of claim 2 , wherein the NLS comprises PKKKRKV.

11 . The method of claim 2 , wherein the Cas9 protein is a nickase comprising D10A mutation.

12 . The method of claim 2 , wherein the Cas9 protein is a nickase comprising at least one of H840A, N854A, or N863A mutation.

13 . The method of claim 2 , wherein the Cas9 protein comprises D10A mutation and at least one of H840A, N854A, or N863A mutation, and wherein the Cas9 protein substantially lacks DNA cleavage activity.

14 . The method of claim 2 , wherein the heterologous protein domain is selected from the group consisting of epitope tags, reporter sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, or nucleic acid binding activity.

15 . The method of claim 2 , wherein the polynucleotide encoding the Cas9 protein is codon-optimized for expression in the eukaryotic cell.

16 . The method of claim 2 , wherein the polynucleotide encoding the Cas9 protein comprises a polyadenylation signal.

17 . The method of claim 2 , wherein the CRISPR-Cas system is comprised in a liposome for delivery.

18 . The method of claim 2 , wherein gene expression associated with the genomic locus of interest is altered in the eukaryotic cell.

19 . A method for editing a eukaryotic cell, comprising delivering an engineered CRISPR-Cas system into the eukaryotic cell, wherein the engineered CRISPR-Cas system comprises:

(a) a Cas9 protein or a polynucleotide encoding the Cas9 protein, wherein the Cas9 protein is S. pyogenes Cas9 and is fused with at least one nuclear localization signal (NLS) and at least one heterologous protein domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, or nucleic acid binding activity; (b) a CRISPR-Cas system chimeric RNA or a polynucleotide encoding the chimeric RNA, wherein the chimeric RNA comprises NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUCA, wherein NNNNNNNNNNNNNNNNNNNN is a guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a genomic locus of interest of the eukaryotic cell; wherein a CRISPR complex comprising the Cas9 protein and the chimeric RNA is formed in the eukaryotic cell, and wherein the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence adjacent to the PAM in the genomic locus of interest of the eukaryotic cell.

20 . A method for editing a eukaryotic cell, comprising delivering an engineered CRISPR-Cas system into the eukaryotic cell, wherein the engineered CRISPR-Cas system comprises:

(a) a polynucleotide encoding a Cas9 protein, wherein the Cas9 protein is S. pyogenes Cas9 and is fused with at least one nuclear localization signal (NLS) and at least one heterologous protein domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, or nucleic acid binding activity, wherein the polynucleotide is codon-optimized for expression in the eukaryotic cell and comprises a polyadenylation signal; (b) a CRISPR-Cas system chimeric RNA comprising NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUCA, wherein NNNNNNNNNNNNNNNNNNNN is a guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a genomic locus of interest of the eukaryotic cell; wherein a CRISPR complex comprising the Cas9 protein and the chimeric RNA is formed in the eukaryotic cell, wherein the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence adjacent to the PAM in the genomic locus of interest, thereby altering gene expression associated with the genomic locus of interest in the eukaryotic cell.

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