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Crispr-cas component systems, methods and compositions for sequence … — The Broad Institute, Inc. (US20250304977A1)

ABSTRACT

Abstract

The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas system.

Description

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. application Ser. No. 17/034,754, filed Sep. 28, 2020, which is a continuation of U.S. application Ser. No. 15/967,495, filed Apr. 30, 2018, which is a continuation of U.S. application Ser. No. 14/105,035, filed Dec. 12, 2013, and which claims priority to U.S. provisional patent applications 61/736,527, filed on Dec. 12, 2012, 61/748,427, filed on Jan. 2, 2013, 61/768,959, filed Feb. 25, 2013, 61/791,409 filed on Mar. 15, 2013, and 61/835,931, filed on Jun. 17, 2013.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

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

Reference is made to U.S. provisional patent applications 61/758,468; 61/769,046; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130, each entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Jan. 30, 2013; Feb. 25, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013 respectively. Reference is also made to U.S. provisional patent applications 61/835,936, 61/836,127, 61/836,101, 61/836,080, 61/836,123 and 61/835,973 each filed Jun. 17, 2013. Reference is also made to U.S. provisional patent application 61/842,322 and U.S. patent application Ser. No. 14/054,414, each having Broad reference BI-2011/008A, entitled CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS filed on Jul. 2, 2013 and Oct. 15, 2013 respectively.

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 Grant No. MH100706, awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Apr. 16, 2025, is named 114203-6221_SL.xml and is 708,469 bytes in size.

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

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

SUMMARY OF THE INVENTION

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

In one aspect, the invention provides a vector system comprising one or more vectors. In some embodiments, the system comprises: (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence; wherein components (a) and (b) are located on the same or different vectors of the system. In some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system comprises the tracr sequence under the control of a third regulatory element, such as a polymerase III promoter. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. Determining optimal alignment is within the purview of one of skill in the art. For example, there are publically and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython and SeqMan. In some embodiments, the CRISPR complex comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR complex in a detectable amount in the nucleus of a eukaryotic cell. Without wishing to be bound by theory, it is believed that a nuclear localization sequence is not necessary for CRISPR complex activity in eukaryotes, but that including such sequences enhances activity of the system, especially as to targeting nucleic acid molecules in the nucleus. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes , or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10 −30 , or between 15-25, or between 15-20 nucleotides in length. In general, and throughout this specification, the term “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 comprises one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and HI 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 EFla 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.).

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

In one aspect, the invention provides a vector comprising a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising one or more nuclear localization sequences. In some embodiments, said regulatory element drives transcription of the CRISPR enzyme in a eukaryotic cell such that said CRISPR enzyme accumulates in a detectable amount in the nucleus of the eukaryotic cell. In some embodiments, the regulatory element is a polymerase II promoter. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.

In one aspect, the invention provides a CRISPR enzyme comprising one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme lacks the ability to cleave one or more strands of a target sequence to which it binds.

In one aspect, the invention provides a eukaryotic host cell comprising (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence. In some embodiments, the host cell comprises components (a) and (b). In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell. In some embodiments, component (a) further comprises the tracr sequence do

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. application Ser. No. 17/034,754, filed Sep. 28, 2020, which is a continuation of U.S. application Ser. No. 15/967,495, filed Apr. 30, 2018, which is a continuation of U.S. application Ser. No. 14/105,035, filed Dec. 12, 2013, and which claims priority to U.S. provisional patent applications 61/736,527, filed on Dec. 12, 2012, 61/748,427, filed on Jan. 2, 2013, 61/768,959, filed Feb. 25, 2013, 61/791,409 filed on Mar. 15, 2013, and 61/835,931, filed on Jun. 17, 2013.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

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

Reference is made to U.S. provisional patent applications 61/758,468; 61/769,046; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130, each entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Jan. 30, 2013; Feb. 25, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013 respectively. Reference is also made to U.S. provisional patent applications 61/835,936, 61/836,127, 61/836,101, 61/836,080, 61/836,123 and 61/835,973 each filed Jun. 17, 2013. Reference is also made to U.S. provisional patent application 61/842,322 and U.S. patent application Ser. No. 14/054,414, each having Broad reference BI-2011/008A, entitled CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS filed on Jul. 2, 2013 and Oct. 15, 2013 respectively.

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 Grant No. MH100706, awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Apr. 16, 2025, is named 114203-6221_SL.xml and is 708,469 bytes in size.

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

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

SUMMARY OF THE INVENTION

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

In one aspect, the invention provides a vector system comprising one or more vectors. In some embodiments, the system comprises: (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence; wherein components (a) and (b) are located on the same or different vectors of the system. In some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system comprises the tracr sequence under the control of a third regulatory element, such as a polymerase III promoter. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. Determining optimal alignment is within the purview of one of skill in the art. For example, there are publically and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython and SeqMan. In some embodiments, the CRISPR complex comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR complex in a detectable amount in the nucleus of a eukaryotic cell. Without wishing to be bound by theory, it is believed that a nuclear localization sequence is not necessary for CRISPR complex activity in eukaryotes, but that including such sequences enhances activity of the system, especially as to targeting nucleic acid molecules in the nucleus. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes , or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10 −30 , or between 15-25, or between 15-20 nucleotides in length. In general, and throughout this specification, the term “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 comprises one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and HI 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 EFla 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.).

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

In one aspect, the invention provides a vector comprising a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising one or more nuclear localization sequences. In some embodiments, said regulatory element drives transcription of the CRISPR enzyme in a eukaryotic cell such that said CRISPR enzyme accumulates in a detectable amount in the nucleus of the eukaryotic cell. In some embodiments, the regulatory element is a polymerase II promoter. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.

In one aspect, the invention provides a CRISPR enzyme comprising one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme lacks the ability to cleave one or more strands of a target sequence to which it binds.

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

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

In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. In some embodiments, said cleavage comprises cleaving one or two strands at the location of the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in decreased transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cell, wherein the one or more vectors drive expression of one or more of: the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence. In some embodiments, said vectors are delivered to the eukaryotic cell in a subject. In some embodiments, said modifying takes place in said eukaryotic cell in a cell culture. In some embodiments, the method further comprises isolating said eukaryotic cell from a subject prior to said modifying. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject.

In one aspect, the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors drive expression of one or more of: the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence.

In one aspect, the invention provides a method of generating a model eukaryotic cell comprising a mutated disease gene. In some embodiments, a disease gene is any gene associated an increase in the risk of having or developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotic cell, wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence; and (b) allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said disease gene, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence, thereby generating a model eukaryotic cell comprising a mutated disease gene. In some embodiments, said cleavage comprises cleaving one or two strands at the location of the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in decreased transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid changes in a protein expression from a gene comprising the target sequence.

In one aspect, the invention provides a method for developing a biologically active agent that modulates a cell signaling event associated with a disease gene. In some embodiments, a disease gene is any gene associated an increase in the risk of having or developing a disease. In some embodiments, the method comprises (a) contacting a test compound with a model cell of any one of the described embodiments; and (b) detecting a change in a readout that is indicative of a reduction or an augmentation of a cell signaling event associated with said mutation in said disease gene, thereby developing said biologically active agent that modulates said cell signaling event associated with said disease gene.

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

In one aspect the invention provides for a method of selecting one or more prokaryotic cell(s) by introducing one or more mutations in a gene in the one or more prokaryotic cell(s), the method comprising: introducing one or more vectors into the prokaryotic cell(s), wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and a editing template; wherein the editing template comprises the one or more mutations that abolish CRISPR enzyme cleavage; allowing homologous recombination of the editing template with the target polynucleotide in the cell(s) to be selected; allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence, wherein binding of the CRISPR complex to the target polynucleotide induces cell death, thereby allowing one or more prokaryotic cell(s) in which one or more mutations have been introduced to be selected. In a preferred embodiment, the CRISPR enzyme is Cas9. In another aspect of the invention the cell to be selected may be a eukaryotic cell. Aspects of the invention allow for selection of specific cells without requiring a selection marker or a two-step process that may include a counter-selection system.

Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 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 model of the CRISPR system. The Cas9 nuclease from Streptococcus pyogenes (yellow) is targeted to genomic DNA by a synthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue) and a scaffold (red). The guide sequence base-pairs with the DNA target (blue), directly upstream of a requisite 5′-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB) ˜3 bp upstream of the PAM (red triangle).

FIGS. 2 A-F . FIG. 2 A shows an exemplary CRISPR system and a possible mechanism of action. FIG. 2 B (left panel) provides an example adaptation of the CRISPR system for expression in eukaryotic cells, and also results of tests assessing nuclear localization and CRISPR activity (right panel). FIG. 2 C illustrates mammalian expression of SpCas9 and SpRNase III driven by the constitutive EFla promoter and tracrRNA and pre-crRNA array (DR-Spacer-DR) driven by the RNA Pol3 promoter U6; and discloses SEQ ID NOS 279-280, respectively, in order of appearance. FIG. 2 D shows results of a surveyor nuclease assay for SpCas9-mediated insertions and deletions. FIG. 2 E is a schematic representation of base pairing between target locus and EMXI-targeting crRNA, as well as an example chromatogram of a micro deletion adjacent to the SpCas9 cleavage site. FIG. 2 E also discloses SEQ ID NOS 281-283, respectively, in order of appearance. FIG. 2 F shows mutated alleles identified from sequencing analysis and discloses SEQ ID NOS 284-288, respectively, in order of appearance.

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

FIGS. 4 A-D show results of an evaluation of SpCas9 specificity for an example target. FIG. 4 A shows effect of single-nucleotide mutations in the guide sequence on protospacer cleavage in the mammalian genome, which was analyzed using a series of EMX1-targeting chimeric crRNAs with single point mutations. FIG. 4 A also discloses SEQ ID NOS 299, 282 and 300-310, respectively, in order of appearance. FIG. 4 B illustrates results of a Surveyor nuclease assay comparing the cleavage efficiency of Cas9 when paired with different mutant chimeric RNAs. FIG. 4 C provides a schematic showing of the design of TALENs targeting EMX1 and also discloses SEQ ID NO: 299. FIG. 4 D shows a Surveyor gel comparing the efficiency of TALEN and Cas9 (n=3).

FIGS. 5 A-G . FIG. 5 A shows an aspartate-to-alanine substitution (D 10A) in the RuvC I catalytic domain of SpCas9 was engineered to convert the nuclease into a nickase. FIG. 5 B shows co-expression of EMX1-targeting chimeric crRNA with SpCas9 produced indels in the target site, whereas co-expression with SpCas9n did not (n=3). FIG. 5 C provides a schematic illustration of the HR strategy, with relative locations of recombination points and primer annealing sequences (arrows). FIG. 5 D shows that SpCas9 and SpCas9n indeed catalyzed integration of the HR template into the EMX1 locus-PCR amplification of the target region followed by restriction digest with HindIII revealed cleavage products corresponding to expected fragment sizes (arrows in restriction fragment length polymorphism gel analysis), with SpCas9 and SpCas9n mediating similar levels of HR efficiencies. FIG. 5 E shows verified HR using Sanger sequencing of genomic amplicons and FIG. 5 E discloses SEQ ID NO: 311. FIG. 5 F shows a schematic design of the crRNA array encoding a pair of EMX1-and PVALB-targeting spacers, and a Surveyor blot showing efficient cleavage at both loci, and also discloses SEQ ID NOS 312-313, respectively, in order of appearance. FIG. 5 G shows targeted deletion of larger genomic regions through concurrent DSBs using spacers against two targets within EMX1 spaced by 119 bp, wherein a 1.6% deletion efficacy (3 out of 182 amplicons) was detected. FIG. 5 G also discloses SEQ ID NOS 314-318, respectively, in order of appearance.

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

FIGS. 7 A-C . FIG. 7 A shows tracrRNA expression constructs and discloses SEQ ID NOS 335-336, respectively, in order of appearance. FIG. 7 B shows a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting in Surveyor assays. FIG. 7 C shows results of a Northern blot analysis of total RNA extracted from 293 FT cells transfected various constructs.

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

FIGS. 9 A-B show exemplary bicistronic expression vectors for expression of CRISPR system elements in eukaryotic cells. FIG. 9 A discloses SEQ ID NOS 337-339, respectively, in order of appearance. FIG. 9 B discloses SEQ ID NOS 340-342, respectively, in order of appearance.

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

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

FIGS. 12 A-C show an exemplary CRISPR system, an example adaptation for expression in eukaryotic cells, and results of tests assessing CRISPR activity. FIG. 12 A provides a Schematic illustration of CRISPR 1 from S. thermophilus LMD-9. FIG. 12 B illustrates the design of an expression system for the S. thermophilus CRISPR system and discloses SEQ ID NOS 346-347, respectively, in order of appearance. An algorithm generating the colors of each base according to its probability of assuming the predicted secondary structure is indicated by a rainbow scale and reproduced in gray scale in FIG. 12 C . FIG. 12 C also discloses SEQ ID NO: 348.

FIGS. 13 A-C show exemplary manipulations of a CRISPR system for targeting of genomic loci in mammalian cells. FIG. 13 A provides a schematic of the human EMXI locus showing the location of five protospacers, indicated by the underlined sequences. FIG. 13 B provides a schematic of the pre-crRNA/trerRNA complex (top), and a schematic of a chimeric RNA design comprising a 20 bp guide sequence in a hairpin structure (bottom). Results of a Surveyor assay is provided in FIG. 13 C . FIG. 13 A also discloses SEQ ID NO: 349. FIG. 13 B also discloses SEQ ID NOS 350-352, respectively, in order of appearance.

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

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

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

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

FIGS. 18 A-C . show exemplary manipulation of a CRISPR system with chimeric RNAs and results of SURVEYOR assays for system activity in eukaryotic cells. FIG. 18 A discloses SEQ ID NO: 364, respectively, in order of appearance and illustrates a schematic of a bicistronic expression vector for chimeric RNA and Cas9. Results of Surveyor assays for Cas9-mediated indels at the human EMXI and PVALB loci are provided in FIG. 18 B and FIG. 18 C , respectively.

FIGS. 19 A-B show a graphical representation of the results of SURVEYOR assays for CRISPR system activity in eukaryotic cells (SEQ ID NOS 365-443, respectively, in order of appearance). Quantification of the results corresponding to FIGS. 18 B and 18 C are illustrated by histogram in FIG. 19 A and FIG. 19 B , respectively.

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

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

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

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

FIGS. 24 A-C show analysis of PAM and seed sequences that eliminate Cas9 cleavage. FIG. 24 A : PCR products with randomized PAM sequences or randomized seed sequences were transformed in crR6 cells (SEQ ID NOS 465-469, respectively, in order of appearance). These cells expressed Cas9 loaded with a crRNA that targeted a chromosomal region of R6 8232.5 cells (highlighted in pink) that is absent from the R6 genome. More than 2×10 5 chloramphenicol-resistant transformants, carrying inactive PAM or seed sequences, were combined for amplification and deep sequencing of the target region. FIG. 24 B : Relative proportion of number of reads after transformation of the random PAM constructs in crR6 cells (compared to number of reads in R6 transformants). The relative abundance for each 3-nucleotide PAM sequence is shown. Severely underrepresented sequences (NGG) are shown in red; partially underrepresented one in orange (NAG). FIG. 24 C : Relative proportion of number of reads after transformation of the random seed sequence constructs in crR6 cells (compared to number of reads in R6 transformants). The relative abundance of each nucleotide for each position of the first 20 nucleotides of the protospacer sequence is shown (SEQ ID NO: 470). High abundance indicates lack of cleavage by Cas9, i.e. a CRISPR inactivating mutation. The grey line shows the level of the WT sequence. The dotted line represents the level above which a mutation significantly disrupts cleavage (See section “Analysis of deep sequencing data” in Example 5).

FIGS. 25 A-F show introduction of single and multiple mutations using the CRISPR system in S. pneumoniae . FIG. 25 A : Nucleotide and amino acid sequences of the wild-type and edited (green nucleotides; underlined amino acid residues) bgaA. The protospacer, PAM and restriction sites are shown (SEQ ID NOS 471-475 and 472, respectively, in order of appearance).

FIG. 25 B : Transformation efficiency of cells transformed with targeting constructs in the presence of an editing template or control. FIG. 25 C : PCR analysis for 8 transformants of each editing experiment followed by digestion with BtgZI (R->A) and Tsel (NE-+AA). Deletion of bgaA was revealed as a smaller PCR product. FIG. 25 D : Miller assay to measure the β-galactosidase activity of WT and edited strains. FIG. 25 E : For a single-step, double deletion the targeting construct contained two spacers (in this case matching srtA and bgaA) and was co-transformed with two different editing templates FIG. 25 F : PCR analysis for 8 transformants to detect deletions in srtA and bgaA loci. 6/8 transformants contained deletions of both genes.

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

FIGS. 27 A-D illustrate genome editing with the CRISPR system in E. coli . FIG. 27 A : A kanamycin-resistant plasmid carrying the CRISPR array (pCRISPR) targeting the gene to edit may be transformed in the HME63 recombineering strain containing a chloramphenicol-resistant plasmid harboring cas9 and tracr (pCas9), together with an oligonucleotide specifying the mutation. FIG. 27 B : A K42T mutation conferring streptomycin resistance was introduced in the rpsl. gene (SEQ ID NOS 480-483, respectively, in order of appearance). FIG. 27 C : Fraction of streptomicyn-resistant (strep?) cfu calculated from total or kanamycin-resistant (kan?) cfu. FIG. 27 D : Fraction of total cells that acquire both the pCRISPR plasmid and the editing oligonucleotide. Co-transformation of the pCRISPR targeting plasmid produced more transformants (t-test, p=0.004). In all cases the values showed the mean+s.d. for three independent experiments.

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

FIGS. 29 A-F provide chromatograms of DNA sequences of edited cells obtained in this study. In all cases the wild-type and mutant protospacer and PAM sequences (or their reverse complement) are indicated. When relevant, the amino acid sequence encoded by the protospacer is provided. For each editing experiment, all strains for which PCR and restriction analysis corroborated the introduction of the desired modification were sequenced. A representative chromatogram is shown. FIG. 29 A : Chromatogram for the introduction of a PAM mutation into the R6 8232.5 target ( FIG. 23 d ) (SEQ ID NOS 484-485, respectively, in order of appearance).

FIG. 29 B : Chromatograms f

CLAIMS

Claims ( 21 )

This listing of claims will replace all prior versions and listings of claims in the application:

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 two or more nuclear localization signals (NLSs); (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 comprises NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUCA.

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

7 . The method of claim 2 , wherein the chimeric RNA is encoded by SEQ ID NO:25.

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

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

10 . The method of claim 2 , wherein the two or more NLSs are independently selected from the group consisting of PKKKRKV, KRPAATKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSP, NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK

11 . The method of claim 10 , wherein at least one of the NLSs comprises PKKKRKV.

12 . The method of claim 2 , wherein the Cas9 protein comprises a mutation of D10A, H840A, N854A, or N863A.

13 . The method of claim 12 , wherein the Cas9 protein is fused with at least one heterologous protein domain.

14 . The method of claim 13 , 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 , further comprising delivering into the eukaryotic cell an exogenous polynucleotide for targeted integration into a DNA break introduced by the CRISPR complex in the genomic locus of interest.

19 . The method of claim 2 , wherein the CRISPR complex cleaves the genomic locus of interest, and wherein the eukaryotic cell is modified with an insertion, deletion, or substitution of one or more nucleotides in the genomic locus of interest.

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 Cas9 protein or a polynucleotide encoding the Cas9 protein, wherein the Cas9 protein is S. pyogenes Cas9 and is fused with two or more nuclear localization signals (NLSs) independently selected from the group consisting of PKKKRKV, KRPAATKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSP, NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK; (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.

21 . 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 two or more nuclear localization signals (NLSs) independently selected from the group consisting of PKKKRKV, KRPAATKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSP, NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK, 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, wherein the CRISPR complex cleaves the genomic locus of interest in the eukaryotic cell.

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Crispr-cas component systems, methods and compositions for sequence manipulation

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( en )

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( 9 )

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( en )

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( 5 )

JP2016505256A

( en )

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( 2 )

KR102955556B1

( en )

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( 3 )

CN114634950A

( en )

AU

( 4 )

AU2013359262C1

( en )

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( 2 )

BR112015013786B1

( en )

CA

( 1 )

CA2894668A1

( en )

DK

( 1 )

DK3252160T3

( en )

WO

( 1 )

WO2014093595A1

( en )

Families Citing this family (951)

* Cited by examiner, † Cited by third party

Publication number

Priority date

Publication date

Assignee

Title

EP2449112A1

( en )

2009-07-01

2012-05-09

Transposagen Biopharmaceuticals, Inc.

Genetically modified rat models for severe combined immunodeficiency (scid)

WO2011140441A2

( en )

2010-05-06

2011-11-10

Children's Hospital Medical Center

Methods and systems for converting precursor cells into intestinal tissues through directed differentiation

US9528124B2

( en )

2013-08-27

2016-12-27

Recombinetics, Inc.

Efficient non-meiotic allele introgression

US10920242B2

( en )

2011-02-25

2021-02-16

Recombinetics, Inc.

Non-meiotic allele introgression

EP2689019A2

( en )

2011-03-23

2014-01-29

Pioneer Hi-Bred International Inc.

Methods for producing a complex transgenic trait locus

WO2013066438A2

( en )

2011-07-22

2013-05-10

President And Fellows Of Harvard College

Evaluation and improvement of nuclease cleavage specificity

EP3604555B1

( en )

2011-10-14

2024-12-25

President and Fellows of Harvard College

Sequencing by structure assembly

WO2014163886A1

( en )

2013-03-12

2014-10-09

President And Fellows Of Harvard College

Method of generating a three-dimensional nucleic acid containing matrix

ES2991004T3

( en )

2011-12-22

2024-12-02

Harvard College

Methods for the detection of analytes

GB201122458D0

( en )

2011-12-30

2012-02-08

Univ Wageningen

Modified cascade ribonucleoproteins and uses thereof

WO2013119602A1

( en )

2012-02-06

2013-08-15

President And Fellows Of Harvard College

Arrdc1-mediated microvesicles (armms) and uses thereof

US9637739B2

( en )

2012-03-20

2017-05-02

Vilnius University

RNA-directed DNA cleavage by the Cas9-crRNA complex

RU2645475C2

( en )

2012-04-25

2018-02-21

Регенерон Фармасьютикалз, Инк.

Nuclease-mediated targeting with large targeting vectors

WO2013163628A2

( en )

2012-04-27

2013-10-31

Duke University

Genetic correction of mutated genes

LT3401400T

( en )

2012-05-25

2019-06-10

The Regents Of The University Of California

Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

US20150225734A1

( en )

2012-06-19

2015-08-13

Regents Of The University Of Minnesota

Gene targeting in plants using dna viruses

US10648001B2

( en )

2012-07-11

2020-05-12

Sangamo Therapeutics, Inc.

Method of treating mucopolysaccharidosis type I or II

CA2875618C

( en )

2012-07-11

2021-04-27

Sangamo Biosciences, Inc.

Methods and compositions for the treatment of lysosomal storage diseases

JP2015527889A

( en )

*

2012-07-25

2015-09-24

ザ ブロード インスティテュート， インコーポレイテッド

Inducible DNA binding protein and genomic disruption tools and their applications

PL4397760T3

( en )

*

2012-10-23

2026-03-30

Toolgen Incorporated

A COMPOSITION FOR CLEFULNING A TARGET DNA COMPRISING A GUIDE RNA SPECIFIC FOR A TARGET DNA AND A NUCLEIC ACID ENCODING A CAS PROTEIN OR A CAS PROTEIN, AND USE THEREOF

WO2014071182A1

( en )

2012-11-01

2014-05-08

Massachusetts Institute Of Technology

Directed evolution of synthetic gene cluster

EP3135765A1

( en )

2012-12-06

2017-03-01

Sigma-Aldrich Co. LLC

Crispr-based genome modification and regulation

WO2014093701A1

( en )

2012-12-12

2014-06-19

The Broad Institute, Inc.

Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof

IL239317B

( en )

2012-12-12

2022-07-01

Broad Inst Inc

Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications

EP2896697B1

( en )

2012-12-12

2015-09-02

The Broad Institute, Inc.

Engineering of systems, methods and optimized guide compositions for sequence manipulation

EP2931898B1

( en )

2012-12-12

2016-03-09

The Broad Institute, Inc.

Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains

US8697359B1

( en )

2012-12-12

2014-04-15

The Broad Institute, Inc.

CRISPR-Cas systems and methods for altering expression of gene products

CN114634950A

( en )

2012-12-12

2022-06-17

布罗德研究所有限公司

CRISPR-CAS component systems, methods, and compositions for sequence manipulation

ES2576126T3

( en )

2012-12-12

2016-07-05

The Broad Institute, Inc.

Modification by genetic technology and optimization of improved enzyme systems, methods and compositions for sequence manipulation

EP3434776A1

( en )

2012-12-12

2019-01-30

The Broad Institute, Inc.

Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof

SG10201912991WA

( en )

*

2012-12-17

2020-03-30

Harvard College

Rna-guided human genome engineering

EP3919505B1

( en )

2013-01-16

2023-08-30

Emory University

Uses of cas9-nucleic acid complexes

US11135273B2

( en )

2013-02-07

2021-10-05

The Rockefeller University

Sequence specific antimicrobials

US10660943B2

( en )

*

2013-02-07

2020-05-26

The Rockefeller University

Sequence specific antimicrobials

SG11201507378UA

( en )

2013-03-14

2015-10-29

Caribou Biosciences Inc

Compositions and methods of nucleic acid-targeting nucleic acids

WO2014204578A1

( en )

2013-06-21

2014-12-24

The General Hospital Corporation

Using rna-guided foki nucleases (rfns) to increase specificity for rna-guided genome editing

US10378027B2

( en )

2013-03-15

2019-08-13

The General Hospital Corporation

RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci

US10760064B2

( en )

2013-03-15

2020-09-01

The General Hospital Corporation

RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci

US20140364333A1

( en )

*

2013-03-15

2014-12-11

President And Fellows Of Harvard College

Methods for Live Imaging of Cells

US9234213B2

( en )

2013-03-15

2016-01-12

System Biosciences, Llc

Compositions and methods directed to CRISPR/Cas genomic engineering systems

US9828582B2

( en )

2013-03-19

2017-11-28

Duke University

Compositions and methods for the induction and tuning of gene expression

JP2016522679A

( en )

2013-04-04

2016-08-04

プレジデント アンド フェローズ オブ ハーバード カレッジ

Therapeutic use of genome editing with the CRISPR / Cas system

DK2986729T3

( en )

2013-04-16

2018-10-29

Regeneron Pharma

TARGETED MODIFICATION OF ROOT THROUGH

ES2670531T3

( en )

*

2013-05-29

2018-05-30

Cellectis S.A.

A method to produce an accurate DNA cleavage using the nickase activity of Cas9

US11414695B2

( en )

2013-05-29

2022-08-16

Agilent Technologies, Inc.

Nucleic acid enrichment using Cas9

SG10201710030QA

( en )

*

2013-06-04

2018-01-30

Harvard College

Rna-guided transcriptional regulation

US20140356956A1

( en )

2013-06-04

2014-12-04

President And Fellows Of Harvard College

RNA-Guided Transcriptional Regulation

ES3029138T3

( en )

*

2013-06-05

2025-06-23

Univ Duke

Rna-guided gene editing and gene regulation

US9982277B2

( en )

2013-06-11

2018-05-29

The Regents Of The University Of California

Methods and compositions for target DNA modification

MX2015017312A

( en )

2013-06-17

2017-04-10

Broad Inst Inc

SUPPLY AND USE OF CRISPR-CAS COMPOSITIONS, VECTORS AND SYSTEMS FOR DIRECTED MODIFICATION AND HEPATIC THERAPY.

CN105793425B

( en )

*

2013-06-17

2021-10-26

布罗德研究所有限公司

Delivery, use and therapeutic applications of CRISPR-CAS systems and compositions for targeting disorders and diseases using viral components

WO2014204724A1

( en )

2013-06-17

2014-12-24

The Broad Institute Inc.

Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation

JP6665088B2

( en )

2013-06-17

2020-03-13

ザ・ブロード・インスティテュート・インコーポレイテッド

Optimized CRISPR-Cas double nickase system, method and composition for sequence manipulation

EP3725885A1

( en )

2013-06-17

2020-10-21

The Broad Institute, Inc.

Functional genomics using crispr-cas systems, compositions methods, screens and applications thereof

JP2016528890A

( en )

2013-07-09

2016-09-23

プレジデント アンド フェローズ オブ ハーバード カレッジ

Therapeutic use of genome editing using the CRISPR / Cas system

US20150044192A1

( en )

2013-08-09

2015-02-12

President And Fellows Of Harvard College

Methods for identifying a target site of a cas9 nuclease

US9359599B2

( en )

2013-08-22

2016-06-07

President And Fellows Of Harvard College

Engineered transcription activator-like effector (TALE) domains and uses thereof

CA3221516A1

( en )

2013-08-22

2015-02-26

E. I. Du Pont De Nemours And Company

Plant genome modification using guide rna/cas endonuclease systems and methods of use

US10760065B2

( en )

2013-09-05

2020-09-01

Massachusetts Institute Of Technology

Tuning microbial populations with programmable nucleases

US9388430B2

( en )

2013-09-06

2016-07-12

President And Fellows Of Harvard College

Cas9-recombinase fusion proteins and uses thereof

US9526784B2

( en )

2013-09-06

2016-12-27

President And Fellows Of Harvard College </td

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