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

Abstract

To provide systems, methods, and compositions for manipulation of sequences and / or activities of target sequences.SOLUTION: 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 for directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR / Cas system.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Incorporation by related applications and references This application claims priority to U.S. Provisional Patent Applications No. 61 / 736,527, No. 61 / 748,427, No. 61 / 768,959, No. 61 / 791,409 and No. 61 / 835,931, all of which have Broad reference numbers BI-2011 / 008 / WSGR no. 44063-701.101, BI-2011 / 008 / WSGR no. 44063-701.102, Broad reference numbers BI-2011 / 008 / VP no. 44790.01.2003, BI-2011 / 008 / VP no. 44790.02.2003 and BI-2011 / 008 / VP no. 44790.03.2003, respectively, all of which are titled SYSTEMS These are METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on December 12, 2012, January 2, 2013, February 25, 2013, March 15, 2013, and June 17, 2013, respectively. The following U.S. Provisional Patent Applications No. 61 / 758,468; No. 61 / 769,046; No. 61 / 802,174; No. 61 / 806,375; No. 61 / 814,263; No. 61 / 819,803 and No. 61 / 828,130 are referenced, with the title ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, respectively, and were filed on January 30, 2013; February 25, 2013; March 15, 2013; March 28, 2013; April 20, 2013; May 6, 2013 and May 28, 2013, respectively. References are also made to U.S. Provisional Patent Applications No. 61 / 835,936, No. 61 / 836,127, No. 61 / 836,101, No. 61 / 836,080, No. 61 / 836,123, and No. 61 / 835,973, which were filed on June 17, 2013.Reference is also made to U.S. Provisional Patent Application No. 61 / 842,322 and U.S. Patent Application No. 14 / 054,414, each having Broad Reference Number BI-2011 / 008A, having the title CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS, and filed on July 2, 2013 and October 15, 2013, respectively.

[0002] The above applications, as well as all documents cited in those applications or during their examination ("application cited documents") and all documents cited or referenced in those application cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in those herein cited documents are hereby incorporated by reference into this specification, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned in this specification or in any document incorporated by reference herein, and can be used in the practice of the present invention. More specifically, all reference documents are incorporated by reference to the extent that each individual document is shown to be incorporated by reference specifically.

[0003] The present invention generally relates to systems, methods, and compositions for controlling gene expression using sequence targeting that can utilize vector systems related to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and its components, such as genome perturbation or gene editing.

[0004] Description of Research Funded by the Federal Government The present invention was made with government support under NIH Pioneer Award DP1MH100706, funded by the National Institutes of Health. The United States Government has certain rights in this invention. BACKGROUND OF THE INVENTION

[0005] Recent advances in genome sequencing technologies and analytical methods have significantly accelerated the ability to classify and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting techniques are necessary to enable the systematic reverse engineering of causal gene mutations by allowing selective perturbation of individual gene elements, and to advance synthetic biology, biotechnology, and pharmaceutical applications. While genome editing technologies, such as designer zinc fingers, transcriptional activator-like effectors (TALEs), or homing meganucleases, are available for producing targeted genome perturbations, there is still a need for novel genome engineering techniques that are inexpensive, easy to set up, scalable, and can easily target multiple locations within the eukaryotic genome. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent No. 4,873,316 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] There is an urgent need for alternative and robust systems and technologies for sequence targeting with diverse applications. This invention addresses this need and provides relevant advantages. CRISPR / Cas or CRISPR-Cas systems (both terms are used interchangeably throughout this application) do not require the generation of customized proteins to target specific sequences, but a single Cas enzyme can be programmed with a short RNA molecule to recognize a specific DNA target; in other words, the Cas enzyme can be recruited to a specific DNA target using the short RNA molecule. The addition of CRISPR-Cas systems to the repertoire of genome sequencing technologies and analytical methods significantly simplifies methodologies and accelerates the skills to classify and map genetic factors associated with a diverse range of biological functions and diseases. To effectively utilize CRISPR-Cas systems for genome editing without adverse effects, it is important to understand the engineering and optimization aspects of those genome engineering tools, which are embodiments of the claimed invention. [Means for solving the problem]

[0008] In one embodiment, the present invention provides a vector system comprising one or more vectors. In some embodiments, the system comprises (a) a first regulatory element operably bound to one or more insertion sites for inserting a tracr mate sequence and one or more guide sequences upstream of the tracr mate sequence (the guide sequences, when expressed, direct sequence-specific binding of the CRISPR complex to a target sequence in a eukaryotic cell, and the CRISPR complex comprises a CRISPR enzyme that complexes with (1) a guide sequence hybridized to the target sequence and (2) a tracr mate sequence hybridized to the tracr sequence); and (b) a second regulatory element operably bound to an enzyme coding sequence encoding the CRISPR enzyme, which comprises a nuclear localization sequence; components (a) and (b) are on the same or different vectors of the system. In some embodiments, component (a) further comprises a tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably bound to a first regulatory element, each of which, when expressed, directs the CRISPR complex to sequence-specific binding to different target sequences in eukaryotic cells. In some embodiments, the system comprises a third regulatory element, e.g., a tracr sequence under the control of a polymerase III promoter. In some embodiments, the tracr sequence exhibits sequence complementarity of at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% along the length of the tracr mate sequence when optimally aligned. Determining optimal alignment is within the scope of the skill of those skilled in the art. For example, there are publicly available and commercially available alignment algorithms and programs, e.g., ClustalW, Smith-Waterman, Bowtie, Geneious, Biopython, and SeqMan in MATLAB. In some embodiments, the CRISPR complex includes one or more nuclear localization sequences of sufficient strength to drive the accumulation of the CRISPR complex in a detectable amount in the nucleus of a eukaryotic cell.While not theoretically constrained, nuclear localization sequences are not required for CRISPR complex activity in eukaryotes, but including such sequences is thought to improve the system's activity, particularly with respect 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 Streptococcus pneumoniae, Streptococcus pyogenes, or S. thermophilus Cas9, and may include mutant Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or orthologue. In some embodiments, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the CRISPR enzyme is directed to cleave one or two strands at the localization of a 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 long, or 10–30, or 15–25, or 15–20 nucleotides long. Generally and throughout this specification, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is bound. Examples of vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends and not containing free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and various other polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be inserted, for example, by standard molecular cloning techniques.Another type of vector is the viral vector, in which a viral-derived DNA or RNA sequence is present in the vector for packaging into a virus (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by viruses for translocation into host cells. Some vectors can self-replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome. Furthermore, some vectors can be directed to the expression of a gene to which they are operatively bound. Such vectors are referred to herein as “expression vectors.” Common expression vectors useful in recombinant DNA technology are often in the form of plasmids.

[0009] A recombinant expression vector may contain the nucleic acid of the present invention in a form suitable for expression of nucleic acid in a host cell, meaning that the recombinant expression vector may be selected based on the host cell to be used for expression and may contain one or more regulatory elements that are operatively bound to the nucleic acid sequence to be expressed. In a recombinant expression vector, "operatively bound" means that the target nucleotide sequence is bound to the regulatory element in such a way that it enables the expression of that nucleotide sequence (e.g., in an in vitro transcription / translation system or, if the vector is introduced into a host cell, in the host cell).

[0010] The term “regulatory element” includes promoters, enhancers, internal ribosome entry sites (IRESs), and other expression regulatory elements (e.g., transcription termination signals, e.g., polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct the constitutive expression of nucleotide sequences in many types of host cells and those that direct the expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory elements). Tissue-specific promoters may primarily direct expression in desired target tissues, e.g., muscle, nerve cells, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). The regulatory elements may also be directed towards time-dependent expression, for example, cell cycle-dependent or developmental stage-dependent expression, which may or may not be tissue- or cell-type specific. In some embodiments, the vector includes one or more polIII promoters (e.g., 1, 2, 3, 4, 5, or more), one or more polII promoters (e.g., 1, 2, 3, 4, 5, or more), one or more polI promoters (e.g., 1, 2, 3, 4, 5, or more), or a combination thereof. Examples of polIII promoters include, but are not limited to, the U6 and H1 promoters.Examples of polII promoters include, but are not limited to, the retroviral Roussarcoma virus (RSV) LTR promoter (sometimes containing an RSV enhancer), the cytomegalovirus (CMV) promoter (sometimes containing a CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. The term “regulatory element” also includes enhancer elements, such as WPRE; CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It is recognized by those skilled in the art that the design of expression vectors may depend on factors such as the selection of host cells to be transformed and the desired expression level. By introducing a vector into host cells, it is possible to produce proteins or peptides, including transcripts, fusion proteins or peptides encoded by the nucleic acids described herein (e.g., clustered equispaced short-chain repeat (CRISPR) transcripts, proteins, enzymes, their mutants, their fusion proteins, etc.).

[0011] Favorable vectors include lentiviruses and adeno-associated viruses, and the type of such vector can also be selected for targeting specific types of cells.

[0012] In one embodiment, the present invention provides a vector comprising a regulatory element operably bound to an enzyme-coding sequence encoding a CRISPR enzyme containing one or more nuclear localization sequences. In some embodiments, the regulatory element drives the transcription of the CRISPR enzyme in eukaryotic cells so that the 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 Streptococcus pneumoniae, Streptococcus pyogenes, or S. thermophilus Cas9, and may include mutant Cas9 derived from these organisms. In some embodiments, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the CRISPR enzyme is directed to cleavage of one or two strands at the localization of a target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.

[0013] In one embodiment, the present invention provides a CRISPR enzyme comprising one or more nuclear localization sequences of sufficient strength to drive the accumulation of the 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 Streptococcus pneumoniae, Streptococcus pyogenes, or S. thermophilus Cas9, and may include mutant Cas9 derived from those organisms. The enzyme may be a Cas9 homolog or orthologue. In some embodiments, the CRISPR enzyme lacks the ability to cleave one or more strands of the target sequence to which it binds.

[0014] In one embodiment, the present invention provides a eukaryotic host cell comprising (a) a first regulatory element operably bound to one or more insertion sites for inserting a tracr mate sequence and one or more guide sequences upstream of the tracr mate sequence (the guide sequences, when expressed, direct sequence-specific binding of the CRISPR complex to a target sequence in a eukaryotic cell, and the CRISPR complex comprises a CRISPR enzyme that complexes with (1) a guide sequence hybridized to the target sequence and (2) a tracr mate sequence hybridized to the tracr sequence); and / or (b) a second regulatory element operably bound to an enzyme-coding sequence encoding the CRISPR enzyme, which comprises a nuclear localization sequence. In some embodiments, the host cell comprises components (a) and (b). In some embodiments, components (a), component (b), or components (a) and (b) are stably integrated into the genome of the host eukaryotic cell. In some embodiments, component (a) further comprises a tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably bound to a first regulatory element, each of which, when expressed, directs the CRISPR complex to sequence-specific binding to different target sequences in a eukaryotic cell. In some embodiments, the eukaryotic host cell further comprises a third regulatory element, e.g., a polymerase III promoter, operably bound to the tracr sequence. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive the accumulation of the 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 Streptococcus pneumoniae, Streptococcus pyogenes, or S. thermophilus Cas9, and may include mutant Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or orthologue. In some embodiments, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the CRISPR enzyme is directed to cleave one or two strands at the localization of a target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides long, or 10–30, or 15–25, or 15–20 nucleotides long. In one embodiment, the present invention provides a non-human eukaryote comprising a eukaryotic host cell according to any of the embodiments described; preferably, a multicellular eukaryote. In another embodiment, the present invention provides a eukaryote comprising a eukaryotic host cell according to any of the embodiments described; preferably, a multicellular eukaryote. In some embodiments of these aspects, the organism may be an animal; for example, a mammal. The organism may also be an arthropod, for example, an insect. The organism may also be a plant. Furthermore, the organism may be a fungus.

[0015] In one embodiment, the present invention provides a kit comprising one or more of the components described herein. In some embodiments, the kit comprises a vector system and instructions for use of the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably bound to one or more insertion sites for inserting a tracr mate sequence and one or more guide sequences upstream of the tracr mate sequence (the guide sequences, when expressed, direct the sequence-specific binding of the CRISPR complex to a target sequence in a eukaryotic cell, and the CRISPR complex comprises a CRISPR enzyme that complexes with (1) a guide sequence hybridized to the target sequence and (2) a tracr mate sequence hybridized to the tracr sequence); and / or (b) a second regulatory element operably bound to an enzyme coding sequence encoding the CRISPR enzyme, which comprises a nuclear localization sequence. In some embodiments, the kit comprises components (a) and (b) on the same or different vectors of the system. In some embodiments, component (a) further comprises a tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably bound to a first regulatory element, each of which, when expressed, directs the CRISPR complex to sequence-specific binding to different target sequences in eukaryotic cells. In some embodiments, the system further comprises a third regulatory element, e.g., a polymerase III promoter, operably bound to the tracr sequence. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive the accumulation of the 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 Streptococcus pneumoniae, Streptococcus pyogenes, or S. thermophilus Cas9, and may include mutant Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or orthologue. In some embodiments, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the CRISPR enzyme is directed to cleave one or two strands at the localization of a target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is the polymerase III promoter. In some embodiments, the second regulatory element is the polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides long, or 10–30, or 15–25, or 15–20 nucleotides long.

[0016] In one embodiment, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises binding a CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide, thereby modifying the target polynucleotide, the CRISPR complex comprising a CRISPR enzyme complexing with a guide sequence which hybridizes to a target sequence within the target polynucleotide, the guide sequence being bound to a tracr mate sequence which then hybridizes to a tracr sequence. In some embodiments, the cleavage comprises the CRISPR enzyme cleaving one or two strands at the localization of the target sequence. In some embodiments, the cleavage results in a reduction in the transcription of the target gene. In some embodiments, the method further comprises repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, the repair resulting in a mutation including the insertion, deletion, or substitution of one or more nucleotides in the target polynucleotide. In some embodiments, the mutation results in a change of one or more amino acids in a protein expressed from the gene containing the target sequence. In some embodiments, the method further comprises delivering one or more vectors to the eukaryotic cells, the one or more vectors driving the expression of one or more CRISPR enzymes, guide sequences bound to tracr mate sequences, and tracr sequences. In some embodiments, the vectors are delivered into the eukaryotic cells in the subject. In some embodiments, the modification is carried out in the eukaryotic cells in a cell culture. In some embodiments, the method further comprises isolating the eukaryotic cells from the subject before the modification. In some embodiments, the method further comprises returning the eukaryotic cells and / or cells derived therefrom to the subject.

[0017] In one embodiment, the present invention provides a method for modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises conjugating a CRISPR complex to a polynucleotide, such that the conjugation results in an increase or decrease in the expression of the polynucleotide; the CRISPR complex comprises a CRISPR enzyme complexing with a guide sequence which hybridizes to a target sequence within the polynucleotide, the guide sequence being bound to a tracr mate sequence which then hybridizes to a tracr sequence. In some embodiments, the method further comprises delivering one or more vectors to the eukaryotic cell, the one or more vectors driving the expression of a CRISPR enzyme, a guide sequence bound to a tracr mate sequence, and one or more tracr sequences.

[0018] In one embodiment, the present invention provides a method for generating a model eukaryotic cell containing a mutated disease gene. In some embodiments, the disease gene is any gene associated with having or having an increased risk of developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotic cell (one or more vectors driving the expression of a CRISPR enzyme, a guide sequence bound to a tracr mate sequence, and one or more tracr sequences) and (b) binding the CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide in the disease gene (the CRISPR complex comprises a CRISPR enzyme complexing with (1) a guide sequence hybridized to a target sequence in the target polynucleotide, and (2) a tracr mate sequence hybridized to a tracr sequence), thereby generating a model eukaryotic cell containing a mutated disease gene. In some embodiments, the cleavage comprises the CRISPR enzyme cleaving one or two strands at the localization of the target sequence. In some embodiments, the cleavage results in a reduction in the transcription of the target gene. In some embodiments, the method further comprises repairing the cleavage target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair results in a mutation including the insertion, deletion, or substitution of one or more nucleotides in the target polynucleotide. In some embodiments, the mutation results in a change of one or more amino acids in a protein expressed from a gene containing the target sequence.

[0019] In one embodiment, the present invention provides a method for developing a bioactive agent that modulates cellular signaling events associated with a disease gene. In some embodiments, the disease gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises (a) contacting a test compound with a model cell of any one of the embodiments described; and (b) detecting a readout change indicating a reduction or increase in cellular signaling events associated with the mutation in the disease gene, thereby developing the bioactive agent that modulates the cellular signaling events associated with the disease gene.

[0020] In one embodiment, the present invention provides a recombinant polynucleotide comprising a guide sequence upstream of a tracr mate sequence, wherein the guide sequence, when expressed, directs the CRISPR complex to sequence-specific binding to a corresponding target sequence present in eukaryotic cells. In some embodiments, the target sequence is a viral sequence present in eukaryotic cells. In some embodiments, the target sequence is a proto-oncogene or oncogene.

[0021] In one embodiment, the present invention provides a method for selecting one or more prokaryotic cells by introducing one or more mutations in the genes of one or more prokaryotic cells, comprising: introducing one or more vectors into the prokaryotic cells (one or more vectors driving the expression of one or more CRISPR enzymes, guide sequences bound to tracr mate sequences, tracr sequences, and editing templates; the editing templates include one or more mutations that halt CRISPR enzyme cleavage); homologous recombination of the editing templates with target polynucleotides in the cells to be selected; and binding the CRISPR complex to the target polynucleotide to cause cleavage of the target polynucleotide in the gene (the CRISPR complex comprises a CRISPR enzyme complexing with (1) a guide sequence hybridized to a target sequence in the target polynucleotide, and (2) a tracr mate sequence hybridized to a tracr sequence, the binding of the CRISPR complex to the target polynucleotide induces cell death), thereby enabling the selection of one or more prokaryotic cells into which one or more mutations have been introduced. In a preferred embodiment, the CRISPR enzyme is Cas9. In another embodiment of the present invention, the cells to be selected may be eukaryotic cells. According to aspects of the present invention, the selection of a specified set of cells is possible without requiring a selection marker or a two-step process that may include a counter-selection system.

[0022] Therefore, the object of the present invention is not to include any already known product, method of producing such product, or method of using such product in the present invention, and therefore the applicants reserve the right to relinquish any already known product, method of producing such product, or method of using such product, and disclose herein this specification. The present invention does not include in the scope any product, method, or method of producing or using such product that does not satisfy the description and enablement requirements of the USPTO (Section 112 of the United States Patent Act) or the EPO (Section 83 of the European Patent Convention), and therefore the applicants reserve the right to relinquish any already described product, method of producing such product, or method of using such product, and disclose herein this specification this specification.

[0023] In this disclosure, and especially in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising", etc. may have the meaning ascribed to them in U.S. patent law; for example, they may mean "includes", "included", "including", etc.; terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, and it is noted that, for example, they allow components not explicitly recited, but exclude components found in the prior art or that affect the basic or novel characteristics of the invention. These and other embodiments are disclosed or are apparent from and are included in the following detailed description.

[0024] The novel features of the invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the 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 to the appended drawings.

Brief Description of the Drawings

[0025] [Figure 1] Shows a schematic model of the CRISPR system. Cas9 nuclease (yellow) from Streptococcus pyogenes is targeted to genomic DNA by a synthetic guide RNA (sgRNA) consisting of a 20nt guide sequence (blue) and a scaffold (red). Base pairing of the guide sequence with the DNA target (blue) immediately upstream of the required 5'-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-strand break (DSB) (red triangle) approximately 3bp upstream of the PAM. [Figure 2A] Shows an exemplary CRISPR system, a possible mechanism of action, an exemplary adaptation of expression in eukaryotic cells, and the results of tests to evaluate nuclear localization and CRISPR activity. [Figure 2B] This paper presents exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations of expression in eukaryotic cells, and results of tests evaluating nuclear localization and CRISPR activity. [Figure 2C] This paper presents exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations of expression in eukaryotic cells, and results of tests evaluating nuclear localization and CRISPR activity. [Figure 2D] This paper presents exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations of expression in eukaryotic cells, and results of tests evaluating nuclear localization and CRISPR activity. [Figure 2E] This paper presents exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations of expression in eukaryotic cells, and results of tests evaluating nuclear localization and CRISPR activity. [Figure 2F] This paper presents exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations of expression in eukaryotic cells, and results of tests evaluating nuclear localization and CRISPR activity. [Figure 3] This paper shows exemplary expression cassettes for the expression of CRISPR system elements in eukaryotic cells, predictive structures of exemplary guide sequences, and CRISPR system activity measured in eukaryotic and prokaryotic cells. [Figure 4A] The results of the evaluation of SpCas9 specificity for exemplary targets are shown. [Figure 4B] The results of the evaluation of SpCas9 specificity for exemplary targets are shown. [Figure 4C] The results of the evaluation of SpCas9 specificity for exemplary targets are shown. [Figure 4D] The results of the evaluation of SpCas9 specificity for exemplary targets are shown. [Figure 5A] The results regarding exemplary vector systems and their use in directing homologous recombination in eukaryotic cells are presented. [Figure 5B] The results regarding exemplary vector systems and their use in directing homologous recombination in eukaryotic cells are presented. [Figure 5C] The results regarding exemplary vector systems and their use in directing homologous recombination in eukaryotic cells are presented. [Figure 5D] The results regarding exemplary vector systems and their use in directing homologous recombination in eukaryotic cells are presented. [Figure 5E] The results regarding exemplary vector systems and their use in directing homologous recombination in eukaryotic cells are presented. [Figure 5F] The results regarding exemplary vector systems and their use in directing homologous recombination in eukaryotic cells are presented. [Figure 5G] The results regarding exemplary vector systems and their use in directing homologous recombination in eukaryotic cells are presented. [Figure 6] This report provides a table of protospacer sequences and summarizes the modification efficiency results for protospacer targets designed based on exemplary Streptococcus pyogenes (S. pyogenes) and S. thermophilus (S. thermophilus) CRISPR systems, and their corresponding PAMs, targeting loci in the human and mouse genomes. Cells were transfused with Cas9 and either precrRNA / tracrRNA or chimeric RNA and analyzed 72 hours after transfusion. Indel percentages were calculated based on Surveyor assay results from the indicated cell lines (N=3 for all protospacer targets; error is standard error (SEM); ND indicates undetectable using the Surveyor assay; NT indicates not tested in this study). [Figure 7A] This paper compares different tracrRNA transcripts for Cas9-mediated gene targeting. [Figure 7B] This paper compares different tracrRNA transcripts for Cas9-mediated gene targeting. [Figure 7C] This paper compares different tracrRNA transcripts for Cas9-mediated gene targeting. [Figure 8] A schematic diagram of a surveyor nuclease assay for detecting microinsertions and deletions induced by double-strand breaks is shown. [Figure 9]This shows an exemplary bicistronic expression vector for the expression of CRISPR system elements in eukaryotic cells. [Figure 10] This shows a bacterial plasmid transformation interference assay, the expression cassette and plasmid used, and the transformation efficiency of the cells used. [Figure 11] Histograms of distances between adjacent Streptococcus pyogenes (S. pyogenes) SF370 locus 1PAM (NGG) (Figure 10A) and S. thermophilus (S. thermophilus) LMD9 locus 2PAM (NNAGAAW) (Figure 10B) in the human genome; and distances for each PAM on a chromosomal (Chr) basis (Figure 10C). [Figure 12] This paper presents exemplary CRISPR systems, exemplary adaptations for expression in eukaryotic cells, and the results of tests evaluating CRISPR activity. [Figure 13] This paper demonstrates exemplary manipulation of the CRISPR system for targeting genomic loci in mammalian cells. [Figure 14] The results of Northern blot analysis of crRNA processing in mammalian cells are shown. [Figure 15] This paper presents exemplary selections of protospacers in the human PVALB and mouse Th gene loci. [Figure 16] This shows exemplary protospacers and corresponding PAM sequence targets of the S. thermophilus CRISPR system in the human EMX1 gene locus. [Figure 17] This provides a table of sequences for primers and probes used in Surveyor, RFLP, genome sequencing, and Northern blot assays. [Figure 18A] This paper presents exemplary manipulations of CRISPR systems containing chimeric RNA and the results of SURVEYOR assays on system activity in eukaryotic cells. [Figure 18B] This paper presents exemplary manipulations of CRISPR systems containing chimeric RNA and the results of SURVEYOR assays on system activity in eukaryotic cells. [Figure 18C]This paper presents exemplary manipulations of CRISPR systems containing chimeric RNA and the results of SURVEYOR assays on system activity in eukaryotic cells. [Figure 19] This graph shows the results of the SURVEYOR assay for CRISPR system activity in eukaryotic cells. [Figure 20] This shows illustrative visualizations of several Streptococcus pyogenes (S. pyogenes) Cas9 target sites in the human genome using the UCSC Genome Browser. [Figure 21] Predicted secondary structures for exemplary chimeric RNAs, including guide sequences, tracr mate sequences, and tracr sequences, are shown. [Figure 22] This shows an exemplary bicistronic expression vector for the expression of CRISPR system elements in eukaryotic cells. [Figure 23] This study demonstrates that Cas9 nuclease activity against endogenous targets can be utilized for genome editing. (a) Concept of genome editing using the CRISPR system. The CRISPR targeting construct aimed to cleave a chromosomal locus and prevented cleavage by co-transforming with an editing template that recombined with the target. Kanamycin-resistant transformants that survived the CRISPR attack contained modifications induced by the editing template, tracr, trans-activated CRISPR RNA; aphA-3, and the kanamycin resistance gene. (b) Transformation of crR6M DNA in R68232.5 cells using no editing template, R6 wild-type srtA, or R6370.1 editing template. Recombination of either R6srtA or R6370.1 prevented Cas9-mediated cleavage. Transformation efficiency was calculated as colony-forming units (cfu) per 1 μg of crR6M DNA; the mean is shown with the standard deviation from at least three independent experiments. PCR analysis was performed on eight clones in each transformation. "Un." indicates the unedited srtA locus of strain R68232.5; "Ed." indicates the editing template. The R68232.5 and R6370.1 targets are distinguished by restriction by EaeI. [Figure 24]Analysis of PAMs and seed sequences that exclude Cas9 cleavage is shown. (a) PCR products containing randomized PAM sequences or randomized seed sequences were transformed into crR6 cells. These cells expressed Cas9 loaded with crRNA targeting the R68232.5 cell chromosomal region absent in the R6 genome (highlighted in pink). More than 2 × 10⁵ chloramphenicol-resistant transformants carrying inactive PAMs or seed sequences were combined for amplification and deep sequencing of the target region. (b) Relative ratio of reads after transformation of random PAM constructs in crR6 cells (compared to reads in R6 transformants). Relative abundance for each 3-nucleotide PAM sequence is shown. Extremely underabundant sequences (NGG) are shown in red; partially underabundant sequences are shown in orange (NAG). (c) Relative ratio of reads after transformation of random seed sequence constructs in crR6 cells (compared to reads in R6 transformants). This shows the relative abundance of each nucleotide at each position in the first 20 nucleotides of the protospacer sequence. High abundances indicate a lack of Cas9 cleavage, i.e., a CRISPR-inactive mutation. The gray line shows the level of the WT sequence. The dotted line represents the level at which the mutation significantly disrupts cleavage (see section "Analysis of Deep Sequencing Data" in Example 5). [Figure 25]This study demonstrates the introduction of single and multiple mutations in Streptococcus pneumoniae using the CRISPR system. (a) Nucleotide and amino acid sequences of wild-type and edited (green nucleotides; underlined amino acid residues) bgaA. Protospacers, PAMs, and restriction sites are shown. (b) Transformation efficiency of cells transformed with the targeting construct in the presence of the editing template or control. (c) PCR analysis of eight transformants for each editing experiment and subsequent digestion with BtgZI (R→A) and TseI (NE→AA). Deletion of bgaA was revealed as a smaller PCR product. (d) Miller assay to measure β-galactosidase activity of WT and edited strains. (e) Due to a single-step double deletion, the targeting construct contained two spacers (in this case, matching srtA and bgaA) and was co-transformed with two different editing templates. (f) PCR analysis of eight transformants to detect deletions at the srtA and bgaA loci. Six out of eight transformants lost both genes. [Figure 26] This study provides an editing-based mechanism using the CRISPR system. (a) A stop codon was introduced into the erythromycin resistance gene ermAM to generate the JEN53 strain. The CRISPR::ermAM(stop) construct could target the stop codon, and the wild-type sequence could be restored by using the ermAM wild-type sequence as an editing template. (b) Mutant and wild-type ermAM sequences. (c) Rate of erythromycin resistance (ermR) cfu calculated from total or kanamycin resistance (kanR) cfu. (d) Rate of total cells acquiring both the CRISPR construct and the editing template. Co-transformation with the CRISPR targeting construct produced more transformants (t-test, p=0.011). In all cases, values ​​represent the mean ± standard deviation for three independent experiments. [Figure 27]This study describes genome editing using the CRISPR system in Escherichia coli (E. coli). (a) A kanamycin-resistant plasmid (pCRISPR) carrying a CRISPR array that targets and edits genes can be used to transform HME63 reproduction strains containing chloramphenicol-resistant plasmid (pCas9) with cas9 and tracr, along with mutation-defining oligonucleotides. (b) The K42T mutation conferring streptomycin resistance was introduced into the rpsL gene. (c) The rate of streptomycin-resistant (strepR) cfus calculated from total or kanamycin-resistant (kanR) cfus. (d) The rate of total cells acquiring both the pCRISPR plasmid and the edited oligonucleotide. Co-transformation with the pCRISPR targeting plasmid produced more transformants (t-test, p=0.004). In all cases, values ​​are shown as the mean ± standard deviation for three independent experiments. [Figure 28]This explains how transformation of crR6 genomic DNA results in editing of targeted gene loci. (a) The crR6 strain was generated by replacing the IS1167 element of Streptococcus pneumoniae (S. pneumoniae) R6 with the CRISPR01 locus of Streptococcus pyogenes (S. pyogenes) SF370. This locus encodes the Cas9 nuclease, a CRISPR array with six spacers, tracrRNA required for crRNA biogenesis, and the proteins Cas1, Cas2, and Csn2, which are not required for targeting. Strain crR6M contains a minimal functional CRISPR system lacking cas1, cas2, and csn2. The aphA-3 gene encodes kanamycin resistance. Protospacers from Streptococcal bacteriophages φ8232.5 and φ370.1 were fused to the chloramphenicol resistance gene (cat) and integrated into the srtA gene of strain R6 to generate strains R68232.5 and R6370.1. (b) Left panel: Transformation of crR6 and crR6M genomic DNA in R68232.5 and R6370.1. Streptomycin resistance genes were also transformed as a control of cellular competence. Right panel: PCR analysis of eight R68232.5 transformants with crR6 genomic DNA. Primers that amplified the srtA locus were used for PCR. Seven out of eight genotyped colonies replaced the R68232.5 srtA locus with a WT locus from crR6 genomic DNA. [Figure 29]This document provides chromatograms of the DNA sequences of the edited cells obtained in this study. In all cases, wild-type and mutant protospacer and PAM sequences (or their inverse complementary strands) are shown. Where relevant, the amino acid sequence encoded by the protospacer is provided. For each editing experiment, all strains that confirmed the introduction of the desired modification by PCR and restriction analysis were sequenced. Representative chromatograms are shown. (a) Chromatogram for the introduction of a PAM mutation into the R68232.5 target (Figure 23d). (b) Chromatogram for the introduction of R>A and NE>AA mutations into β-galactosidase (bgaA) (Figure 25c). (c) Chromatogram for the introduction of a 6664bp deletion in the bgaA ORF (Figures 25c and 25f). The dotted line indicates the deletion limit. (d) Chromatogram for the introduction of a 729bp deletion in the srtA ORF (Figure 25f). The dotted line indicates the deletion limit. (e) Chromatogram of early stop codon generation in ermAM (Figure 33). (f) rpsL editing in Escherichia coli (Figure 27). [Figure 30] This study describes CRISPR immunity against random Streptococcus pneumoniae (S. pneumoniae) targets containing different PAMs. (a) Locations of 10 random targets on the S. pneumoniae (S. pneumoniae) R6 genome. The selected targets have different PAMs and are present on both strands. (b) Spacers corresponding to the targets were cloned in a minimal CRISPR array on plasmid pLZ12 and transformed into strain crR6Rc, supplying the processing and targeting mechanisms in trans. (c) Transformation efficiency of different plasmids in strains R6 and crR6Rc. No colonies were recovered for transformation of pDB99-108 (T1-T10) in crR6Rc. The dotted line represents the detection limit of the assay. [Figure 31]This paper provides a general scheme for targeted genome editing. To facilitate targeted genome editing, crR6M was further engineered to contain only one repeat of tracrRNA, Cas9, and CRISPR arrays, followed by a kanamycin resistance marker (aphA-3), generating the strain crR6Rk. DNA from this strain was used as a template for PCR, along with primers designed to introduce a novel spacer (green box indicated by N). Left and right PCRs were assembled using the Gibson method to create a targeting construct. Both the targeting and editing constructs were then transformed into strain crR6Rc, which is equivalent to crR6Rk, but the kanamycin resistance marker was replaced with a chloramphenicol resistance marker (cat). Approximately 90% of the kanamycin-resistant transformants contained the desired mutation. [Figure 32] This section describes the distribution of distances between PAMs. NGG and CCN are considered valid PAMs. Data are shown for the *Streptococcus pneumoniae* (S. pneumoniae) R6 genome and random sequences with identical length and GC content (39.7%). The dotted line represents the average distance (12) between PAMs in the R6 genome. [Figure 33]This paper describes CRISPR-mediated editing of the ermAM locus using genomic DNA as a targeting construct. Because genomic DNA is used as the targeting construct, it is necessary to avoid CRISPR autoimmunity; therefore, a spacer for a sequence not present in the chromosome must be used (in this case, the ermAM erythromycin resistance gene). (a) Nucleotide and amino acid sequences of the wild-type and mutant (red text) ermAM genes. Protospacer and PAM sequences are shown. (b) Schematic diagram of CRISPR-mediated editing of the ermAM locus using genomic DNA. Constructs carrying the ermAM targeting spacer (blue box) were prepared by PCR and Gibson assembly and transformed into strain crR6Rc to generate strain JEN37. Then, the genomic DNA of JEN37 was used as the targeting construct and co-transformed with the editing template into strain JEN38, in which the srtA gene was replaced with a wild-type copy of ermAM. Kanamycin-resistant transformants contain the edited genotype (JEN43). (c) Number of kanamycin-resistant cells obtained after targeting and co-transformation with edited or control templates. 5.4 × 10³ cfu / ml was obtained in the presence of the control template, and 4.3 × 10⁵ cfu / ml was obtained when using the edited template. This difference indicates an editing efficiency of approximately 99% [(4.3 × 10⁵ - 5.4 × 10³) / 4.3 × 10⁵]. (d) To confirm the presence of edited cells, seven kanamycin-resistant clones and JEN38 were streaked on erythromycin-containing (erm+) or erythromycin-absent (erm-) agar plates. Only the positive control showed resistance to erythromycin. The ermAMmut genotype of one of these transformants was also confirmed by DNA sequencing (Figure 29e). [Figure 34]This document describes the sequential introduction of mutations by CRISPR-mediated genome editing. (a) Schematic diagram of the sequential introduction of mutations by CRISPR-mediated genome editing. First, R6 is engineered to produce crR6Rk. crR6Rk is co-transformed with an editing construct for ΔsrtA in-frame deletion, along with an srtA targeting construct fused to cat for chloramphenicol selection of the edited cells. The strain crR6ΔsrtA is produced by chloramphenicol-based selection. Subsequently, the ΔsrtA strain is co-transformed with an editing construct containing ΔbgaA in-frame deletion, along with an aphA-3 targeting construct fused to aphA-3 for kanamycin selection of the edited cells. Finally, the engineered CRISPR locus can be erased from the chromosome by co-transforming with a plasmid (pDB97) carrying R6 DNA and a bgaA protospacer containing the wild-type IS1167 locus, and then selecting based on spectinomycin. (b) PCR analysis of eight chloramphenicol (Cam)-resistant transformants to detect deletion of the srtA gene locus. (c) β-galactosidase activity measured by Miller assay. In Streptococcus pneumoniae, this enzyme is fixed to the cell wall by saltase A. Deletion of the srtA gene results in the release of β-galactosidase into the supernatant. The ΔbgaA mutant shows no activity. (d) PCR analysis of eight spectinomycin (Spec)-resistant transformants to detect replacement of the CRISPR gene locus by wild-type IS1167. [Figure 35]This paper describes the background mutation frequency of CRISPR in Streptococcus pneumoniae. (a) Transformation of JEN53 with or without the ermAM editing template using CRISPR::φ or CRISPR::erm(stop) targeting constructs. The difference in kanRCFU between CRISPR::φ and CRISPR::erm(stop) indicates that Cas9 cleavage kills non-edited cells. Mutants that escape CRISPR interference in the absence of the editing template are observed at a frequency of 3 × 10⁻³. (b) PCR analysis of the escaper CRISPR locus shows that 7 out of 8 have spacer deletions. (c) Escaper #2 carries a point mutation in cas9. [Figure 36] This document describes how essential elements of the CRISPR locus 1 in Streptococcus pyogenes (S. pyogenes) can be reconstituted in Escherichia coli (E. coli) using pCas9. The plasmid contained tracrRNA, Cas9, and a leader sequence to drive the crRNA array. The pCRISPR plasmid contained only the leader and array. Spacers can be inserted into the crRNA array between BsaI sites using annealed oligonucleotides. Oligonucleotide designs are shown below. pCas9 carries chloramphenicol resistance (CmR) and is based on a low-copy pACYC184 plasmid backbone. pCRISPR is based on a high-copy-number pZE21 plasmid. Two plasmids were required because the pCRISPR plasmid, containing a spacer targeting the E. coli chromosome, cannot be constructed using this organism as a cloning host if Cas9 is also present (it would kill the host). [Figure 37]This report describes CRISPR-directed editing in *E. coli* MG1655. An oligonucleotide (W542) carrying a point mutation conferring streptomycin resistance and terminating CRISPR immunity was co-transformed into wild-type *E. coli* strain MG1655 containing pCas9, along with either a plasmid targeting rpsL (pCRISPR::rpsL) or a control plasmid (pCRISPR::φ). Transformants were selected on a medium containing either streptomycin or kanamycin. The dotted line indicates the detection limit of the transformation assay. [Figure 38] This paper describes the background mutation frequency of CRISPR in *E. coli* HME63. (a) Transformation of HME63 competent cells with pCRISPR::φ or pCRISPR::rpsL plasmids. Mutants escaping CRISPR interference were observed at a frequency of 2.6 × 10⁻⁴. (b) Amplification of the escaper CRISPR array showed that 8 out of 8 lacked the spacer. [Figure 39A] This circular diagram shows the phylogenetic analysis revealing five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). [Figure 39B] This circular diagram shows the phylogenetic analysis revealing five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). [Figure 39C] This circular diagram shows the phylogenetic analysis revealing five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). [Figure 39D] This circular diagram shows the phylogenetic analysis revealing five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). [Figure 40A]This shows a linear representation of phylogenetic analysis that identifies five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). [Figure 40B] This shows a linear representation of phylogenetic analysis that identifies five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). [Figure 40C] This shows a linear representation of phylogenetic analysis that identifies five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). [Figure 40D] This shows a linear representation of phylogenetic analysis that identifies five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). [Figure 40E] This shows a linear representation of phylogenetic analysis that identifies five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). [Figure 40F] This shows a linear representation of phylogenetic analysis that identifies five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). [Figure 41A] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41B] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41C] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41D] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41E] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41F] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41G] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41H] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41I] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41J] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41K] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41L] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 41M] This shows the sequence in which the mutation site is localized within the SpCas9 gene. [Figure 42] This diagram shows a schematic construct in which a transcriptional activation domain (VP64) is fused to Cas9, which has two mutations (D10 and H840) in its catalytic domain. [Figure 43A] This shows genome editing via homologous recombination. (a) Schematic diagram of SpCas9 nickase with a D10A mutation in the RuvC I catalytic domain. (b) Schematic diagram representing homologous recombination (HR) at the human EMX1 locus using either sense or antisense single-stranded oligonucleotide as a repair template. The red arrow at the top indicates the sgRNA cleavage site; PCR primers for genotyping (Table J and K) are shown as arrows in the right panel. (c) Sequence of the region modified by HR. d. SURVEYOR assay (n=3) for wild-type (wt) and nickase (D10A) SpCas9-mediated indels at the EMX target 1 locus. Arrows indicate the location of the predicted fragment size. [Figure 43B]This shows genome editing via homologous recombination. (a) Schematic diagram of SpCas9 nickase with a D10A mutation in the RuvC I catalytic domain. (b) Schematic diagram representing homologous recombination (HR) at the human EMX1 locus using either sense or antisense single-stranded oligonucleotide as a repair template. The red arrow at the top indicates the sgRNA cleavage site; PCR primers for genotyping (Table J and K) are shown as arrows in the right panel. (c) Sequence of the region modified by HR. d. SURVEYOR assay (n=3) for wild-type (wt) and nickase (D10A) SpCas9-mediated indels at the EMX target 1 locus. Arrows indicate the location of the predicted fragment size. [Figure 43C] This shows genome editing via homologous recombination. (a) Schematic diagram of SpCas9 nickase with a D10A mutation in the RuvC I catalytic domain. (b) Schematic diagram representing homologous recombination (HR) at the human EMX1 locus using either sense or antisense single-stranded oligonucleotide as a repair template. The red arrow at the top indicates the sgRNA cleavage site; PCR primers for genotyping (Table J and K) are shown as arrows in the right panel. (c) Sequence of the region modified by HR. d. SURVEYOR assay (n=3) for wild-type (wt) and nickase (D10A) SpCas9-mediated indels at the EMX target 1 locus. Arrows indicate the location of the predicted fragment size. [Figure 43D] This shows genome editing via homologous recombination. (a) Schematic diagram of SpCas9 nickase with a D10A mutation in the RuvC I catalytic domain. (b) Schematic diagram representing homologous recombination (HR) at the human EMX1 locus using either sense or antisense single-stranded oligonucleotide as a repair template. The red arrow at the top indicates the sgRNA cleavage site; PCR primers for genotyping (Table J and K) are shown as arrows in the right panel. (c) Sequence of the region modified by HR. d. SURVEYOR assay (n=3) for wild-type (wt) and nickase (D10A) SpCas9-mediated indels at the EMX target 1 locus. Arrows indicate the location of the predicted fragment size. [Figure 44A] This document demonstrates a single-vector design for SpCas9. [Figure 44B] This document presents a single-vector design for SpCas9. [Figure 45] This paper quantifies the NLS-Csn1 constructs NLS-Csn1, Csn1, Csn1-NLS, NLS-Csn1-NLS, NLS-Csn1-GFP-NLS, and UnTFN cleavage. [Figure 46] The exponential frequencies of NLS-Cas9, Cas9, Cas9-NLS, and NLS-Cas9-NLS are shown. [Figure 47] This gel demonstrates that SpCas9 with nickase mutations does not induce double-strand breaks (individually). [Figure 48] This study presents the design of the oligoDNA used as the homologous recombination (HR) template in this experiment, as well as a comparison of the HR efficiency induced by different combinations of Cas9 protein and HR template. [Figure 49A] Conditional Cas9 and Rosa26 targeting vector maps are shown. [Figure 49B] Constitutive Cas9 and Rosa26 targeting vector maps are shown. [Figure 50A] Figures 49A and 49B show the arrangement of each element present in the vector map. [Figure 50B] Figures 49A and 49B show the arrangement of each element present in the vector map. [Figure 50C] Figures 49A and 49B show the arrangement of each element present in the vector map. [Figure 50D] Figures 49A and 49B show the arrangement of each element present in the vector map. [Figure 50E] Figures 49A and 49B show the arrangement of each element present in the vector map. [Figure 50F] Figures 49A and 49B show the arrangement of each element present in the vector map. [Figure 50G]Figures 49A and 49B show the arrangement of each element present in the vector map. [Figure 50H] Figures 49A and 49B show the arrangement of each element present in the vector map. [Figure 51] This shows schematic diagrams of key elements in constructive and conditional Cas9 structures. [Figure 52] This paper demonstrates the functional validation of the expression of constitutive and conditional Cas9 constructs. [Figure 53] This shows the verification of Cas9 nuclease activity using Surveyor. [Figure 54] This shows the quantitative analysis of Cas9 nuclease activity. [Figure 55] This document outlines the structural design and homologous recombination (HR) policies. [Figure 56] The results of genomic PCR genotyping for constitutive (right) and conditional (left) constructs at two different gel exposure times (3 minutes for the top row and 1 minute for the bottom row) are shown. [Figure 57] This shows Cas9 activation during mesentery estrogen (mESC). [Figure 58] A schematic diagram of the strategy used to mediate NHEJ-mediated gene knockout using a nickase version of Cas9 along with two guide RNAs is shown. [Figure 59] This demonstrates how DNA double-strand break (DSB) repair facilitates gene editing. In the error-prone non-homologous end joining (NHEJ) pathway, the ends of a DSB are processed by endogenous DNA repair mechanisms and rejoined together, which can result in random insertion / deletion (indel) mutations at the junction. Indel mutations occurring within the coding region of a gene can result in frameshifts and premature stop codons, leading to gene knockout. Alternatively, homologous recombination repair (HDR) pathways can be utilized, providing repair templates in the form of plasmids or single-stranded oligodeoxynucleotides (ssODNs) to enable high-fidelity and precise editing. [Figure 60]The experimental timeline and overview are shown, covering the steps of reagent design, construction, validation, and cell line proliferation. Custom sgRNAs (light blue bars) and genotyping primers for each target are designed in silico via the applicant's online design tool (available at genome-engineering.org / tools). The sgRNA expression vector is then cloned into a Cas9-containing plasmid (PX330) and validated via DNA sequencing. The completed plasmid (pCRISPR) and an optional repair template to promote homologous recombination repair are then transfected into cells and assayed for their ability to mediate targeted cleavage. Finally, the transfected cells can be cloned to obtain an isogenic cell line with the specified mutations. [Figure 61]Target selection and reagent preparation are shown. (a) For Streptococcus pyogenes (S. pyogenes) Cas9, the 20 bp target (highlighted in blue) must be followed by a 5'-NGG that can occur on any strand of genomic DNA. The applicants recommend using the online tools described in this protocol to assist in target selection (www.genome-engineering.org / tools). (b) Schematic diagram of simultaneous translocation of a Cas9 expression plasmid (PX165) and a PCR-amplified U6-driven sgRNA expression cassette. Using a U6 promoter-containing PCR template and fixed forward primer (U6Fwd), sgRNA coding DNA can be added to a U6 reverse primer (U6Rev) and synthesized as an extended DNA oligo (Ultramer oligo from IDT). Note that the guide sequence in U6Rev (N in blue) is the reverse complementary strand of the 5'-NGG flanking target sequence. (c) Schematic diagram of scarless cloning of guide sequence oligo into a plasmid (PX330) containing Cas9 and sgRNA scaffold. The guide oligo (blue N) contains an overhang for ligation into a pair of BbsI sites on PS330, with top and bottom strand orientation matching that of the genomic target (i.e., the top oligo is a 20 bp sequence preceding 5'-NGG in genomic DNA). Digestion of PX330 by BbsI allows replacement of the type IIs restriction site (blue box) by direct insertion of the annealed oligo. Note that the additional G is placed before the first base of the guide sequence. We have found that the additional G before the guide sequence does not adversely affect targeting efficiency. If the optimal 20 nt guide sequence does not start with guanine, the additional guanine ensures that the sgRNA is efficiently transcribed by the U6 promoter, which preferentially starts with guanine in the first base of the transcript. [Figure 62]Prediction results for multiple NHEJ are shown. (a) Schematic diagram of the SURVEYOR assay used to determine the indel rate. First, genomic DNA from a heterogeneous population of Cas9-targeted cells is amplified by PCR. Then, the amplicons are slowly reannealed to generate heteroduplexes. The reannealed heteroduplexes are cleaved by SURVEYOR nuclease, while the homoduplexes are left intact. Cas9-mediated cleavage efficiency (% indels) is calculated based on the rate of cleaved DNA determined by the combined intensity of the gel bands. (b) Two sgRNAs (orange and blue bars) are designed to target the human GRIN2B and DYRK1A loci. The SURVEYOR gel shows modifications at both loci in the transfected cells. Colored arrows indicate the predicted fragment size for each locus. (c) A pair of sgRNAs (light blue and green bars) are designed to excise an exon (dark blue) from the human EMX1 locus. The target sequence and PAM (red) are shown in their respective colors, and the cleavage site is indicated by a red triangle. The predicted junction is shown below. Individual clones isolated from cell populations transfected with sgRNA3, 4, or both were assayed by PCR (OUT Fwd, OUT Rev) to reflect a deletion of approximately 270 bp. Representative clones with no modification (12 / 23), monoallelic (10 / 23), and biallelic (1 / 23) modifications are shown. Inversion events were screened using IN Fwd and IN Rev primers (Figure 6d). (d) Quantification of clonal lines deleting the EMX1 exon. Variable-sized deletions around a single EMX1 exon were mediated using two pairs of sgRNAs (3.1, 3.2 left-side flanking sgRNA; 4.1, 4.2 right-side flanking sgRNA). Transfected cells were cloned and grown for genotyping analysis of deletion and inversion events. Of the 105 clones, 51 (49%) and 11 (10%) carrying heterozygous and homozygous deletions, respectively, were screened. Since zygosity can be variable, the estimated deletion size is given. [Figure 63]We demonstrate the application of ssODN and targeting vectors to mediate HR using both wild-type and nickase mutant Cas9 in HEK293FT and HUES9 cells, with efficiencies ranging from 1.0 to 27%. [Figure 64] A schematic diagram of a PCR-based method for rapid and efficient CRISPR targeting in mammalian cells is shown. A plasmid containing the human RNA polymerase III promoter U6 is PCR-amplified using a U6-specific forward primer, a reverse primer carrying a portion of the inverse complementary strand of the U6 promoter, an sgRNA(+85) scaffold with a guide sequence, and seven T nucleotides for transcription termination. The resulting PCR product is purified and co-delivered with a plasmid carrying Cas9 driven by the CBh promoter. [Figure 65] The SURVEYOR Mutation Detection Kit results from Transgenomics for each gRNA and its control are shown. A positive SURVEYOR result consists of one large band corresponding to genomic PCR and two smaller bands, which are the product of the SURVEYOR nuclease creating double-strand breaks at the mutation site. Each gRNA was validated in the mouse cell line Neuro-N2a by liposome transient simultaneous translocation with hSpCas9. Genomic DNA was purified 72 hours after translocation using QuickExtract DNA from Epicentre. PCR was performed to amplify the target locus. [Figure 66] The Surveyor results for 38 surviving pups (lanes 1-38), one dead pup (lane 39), and one wild-type pup for comparison (lane 40) are shown. gRNA Chd8.2 was injected into pups 1-19, and gRNA Chd8.3 was injected into pups 20-38. Of the 38 surviving pups, 13 were positive for mutations. The one dead pup also had mutations. No mutations were detected in the wild-type samples. Genomic PCR sequencing was consistent with the findings of the Surveyor assay. [Figure 67]The designs of different Cas9 NLS constructs are shown. All Cas9s were human codon-optimized versions of SpCas9. The NLS sequence was ligated to the cas9 gene either at the N-terminus or C-terminus. All Cas9 variants with different NLS designs were cloned into a skeletal vector containing them so that they were driven by the EF1a promoter. A chimeric RNA targeting the human EMX1 locus, driven by the U6 promoter, was present on the same vector, forming a two-component system together. [Figure 68] This shows the efficiency of genome cleavage induced by Cas9 variants carrying different NLS designs. The percentages represent the portion of human EMX1 genomic DNA cleaved by each construct. All experiments were performed from three biological copies, n=3, and the error is shown as the standard error (SEM). [Figure 69A] This study demonstrates a CRISPR-TF (transcription factor) design with transcriptional activation activity. A chimeric RNA is expressed via the U6 promoter, while a human codon-optimized double mutant version of the Cas9 protein (hSpCas9m), operably bound to three NLS and VP64 functional domains, is expressed via the EF1a promoter. The double mutants D10A and H840A render the Cas9 protein unable to induce any cleavage, but its ability to bind to target DNA when guided by the chimeric RNA is maintained. [Figure 69B]This study demonstrates transcriptional activation of the human SOX2 gene by the CRISPR-TF system (chimeric RNA and Cas9-NLS-VP64 fusion protein). 293FT cells were transfused with a plasmid containing two components: (1) a different chimeric RNA driven by U6 targeting a 20 bp sequence within or around the human SOX2 genomic locus, and (2) an hSpCas9m (double mutant)-NLS-VP64 fusion protein driven by EF1a. 96 hours after transfusion, 293FT cells were harvested, and the level of activation was measured by inducing mRNA expression using a qRT-PCR assay. All expression levels were normalized against a control group (gray bars) representing results from cells transfused with a CRISPR-TF backbone plasmid without chimeric RNA. The qRT-PCR probe used for SOX2 mRNA detection was the Taqman Human Gene Expression Assay (Life Technologies). All experiments represent data from three biological replicas, n=3, and the error bars indicate the standard error (SEM). [Figure 70] This document demonstrates the NLS architecture optimization for SpCas9. [Figure 71] A QQ plot for the NGGNN sequence is shown. [Figure 72] The histogram of data density is shown along with the fitted normal distribution (black line) and the 0.99 quantile (dotted line). [Figure 73] This shows RNA-guided repression of bgaA expression by dgRNA::cas9**. a. The Cas9 protein binds to tracrRNA and precursor CRISPR RNA, which are processed by RNase III to form crRNA. The crRNA directs Cas9 to the bgaA promoter, repressing transcription. b. Represents the targets used to direct Cas9** to the bgaA promoter. Putative -35, -10, and bgaA start codons are shown in bold. c. Beta-galactosidase activity measured by Miller assay in the absence of targeting and for four different targets. [Figure 74] This shows the characterization of Cas9**-mediated repression. a. The gfpmut2 gene and its promoter, along with the locations of the different target sites used in this study, including the -35 and -10 signals. b. Relative fluorescence when targeting the coding strand. c. Relative fluorescence when targeting the non-coding strand. d. Northern blots using probes B477 and B478 on RNA extracted from T5, T10, B10, or control strains without targets. e. Effect of mutations in the number of additions at the 5' end of crRNA in B1, T5, and B10. [Modes for carrying out the invention]

[0026] The drawings in this specification are for illustrative purposes only and are not necessarily drawn to a specific scale.

[0027] The terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” “nucleic acid,” and “oligonucleotide” are used interchangeably. These refer to polymeric forms of nucleotides, deoxyribonucleotides, or ribonucleotides of any length, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (gene loci) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, e.g., methylated nucleotides or nucleotide analogs. Modifications to the nucleotide structure, if present, can be given before or after the assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component.

[0028] In aspects of the present invention, the terms “chimeric RNA,” “chimeric guide RNA,” “guide RNA,” “single guide RNA,” and “synthetic guide RNA” are used interchangeably and refer to a polynucleotide sequence comprising a guide sequence, a tracr sequence, and a tracr mate sequence. The term “guide sequence” refers to a sequence of approximately 20 bp within the guide RNA that defines the target site and can be used interchangeably with the terms “guide” or “spacer.” The term “tracr mate sequence” can also be used interchangeably with the term “direct repeat.”

[0029] As used herein, the term “wild type” is a term understood by those skilled in the art and means a typical form of an organism, strain, gene, or characteristic as it occurs in nature, distinguished from mutant or variant forms.

[0030] As used herein, the term "variant" should be interpreted as meaning a presentation of quality that has a pattern that deviates from that which occurs in the natural state.

[0031] The terms “not naturally occurring” or “engineered” are used interchangeably and indicate artificial involvement. When these terms refer to nucleic acid molecules or polypeptides, they mean that the nucleic acid molecules or polypeptides do not contain, at least substantially, at least one other component that they naturally associate with or are found in nature.

[0032] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either through classical Watson-Crick base pairing or other non-classical types. The complementarity percentage indicates the proportion of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (for example, 5, 6, 7, 8, 9, and 10 out of 10 have 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). "Perfectly complementary" means that all consecutive residues in a nucleic acid sequence can form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. As used herein, “substantially complementary” means a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% of a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or two nucleic acids that hybridize under stringent conditions.

[0033] As used herein, “stringent conditions” for hybridization refer to conditions under which a nucleic acid complementary to the target sequence predominantly hybridizes with the target sequence and substantially does not hybridize with the non-target sequence. Stringent conditions are generally sequence-dependent and vary depending on numerous factors. Generally, longer sequences have higher temperatures at which they specifically hybridize with their target sequence. Non-limiting examples of stringent conditions are detailed in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology—Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, NY.

[0034] Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur through Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific manner. The complex may consist of two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybriding strand, or any combination thereof. Hybridization reactions may constitute a step in a broader process, such as the initiation of PCR or enzymatic cleavage of polynucleotides. A sequence that can hybridize with a given sequence is referred to as a "complementary strand" of the given sequence.

[0035] As used herein, “expression” refers to the process by which polynucleotides are transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and the polypeptides they encode can be collectively referred to as “gene products.” If the polynucleotides originate from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0036] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, and may contain modified amino acids, which may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have undergone modifications, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation, such as conjugation with a labeling component. The term “amino acid” as used herein includes natural and / or unnatural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptide mimetic compounds.

[0037] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, mice, monkeys, humans, domestic animals, athletic animals, and pets. Tissues, cells, and their offspring of biological entities obtained in vivo or cultured in vitro are also included.

[0038] The terms "therapeutic agent," "therapeutic drug," or "treatment agent" are used interchangeably to refer to molecules or compounds that, when administered to a subject, impart several beneficial effects. These beneficial effects include the availability of diagnostic measurements; improvement of a disease, symptom, disorder, or pathological condition; reduction of a disease, symptom, disorder, or pathological condition or prevention of its onset; and, generally, neutralization of a disease, symptom, disorder, or pathological condition.

[0039] As used herein, “treatment,” “to treat,” “to alleviate,” or “to improve” are interchangeable. These terms refer to an approach to obtain a benefit or desired outcome, for example, but not limited to, a therapeutic benefit and / or a preventive benefit. A therapeutic benefit means any treatment-related improvement or effect on one or more diseases, conditions, or symptoms during treatment. A preventive benefit means that a composition may be administered to subjects at risk of developing a particular disease, condition, or symptom, or to subjects reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptoms have not previously manifested.

[0040] The term “effective dose” or “therapeutic effective dose” refers to the amount of drug sufficient to produce a benefit or desired outcome. The therapeutic effective dose may vary depending on one or more of the subject and condition being treated, the subject’s weight and age, the severity of the condition, and the mode of administration, which can be readily determined by those skilled in the art. This term also applies to the dose that provides an image for detection by any of the imaging methods described herein. The prescribed dose may vary depending on one or more of the specific drug selected, the administration regimen to be followed, whether or not it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system on which it is carried.

[0041] The implementation of this invention will, unless otherwise specified, utilize conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the scope of the skill of those skilled in the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FMAusubel, et al. eds., (1987)); series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GRTaylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RIFreshney, ed. (1987)).

[0042] Some aspects of the present invention relate to vector systems comprising one or more vectors, or to the vectors themselves. The vectors can be designed for the expression of CRISPR transcripts (e.g., nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells, e.g., Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, *Gene Expression Technology: Methods in Enzymology* 185, Academic Press, San Diego, Calif. (1990). Alternatively, recombinant expression vectors can be transcribed and translated in vitro, for example, using a T7 promoter regulatory sequence and T7 polymerase.

[0043] Vectors can be introduced into prokaryotes and grown within them. In some embodiments, prokaryotes are used to amplify copies of a vector to be introduced into eukaryotic cells, or as intermediate vectors in the production of vectors to be introduced into eukaryotic cells (e.g., amplifying plasmids as part of a viral vector packaging system). In some embodiments, prokaryotes are used to amplify copies of a vector to express one or more nucleic acids, providing, for example, a resource of one or more proteins for delivery to a host cell or host organism. Protein expression in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inductive promoters directed towards the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to the protein they encode, for example, to the amino terminus of a recombinant protein. Such fusion vectors can serve one or more purposes, for example, (i) increased expression of recombinant proteins; (ii) increased solubility of recombinant proteins; and (iii) assistance in the purification of recombinant proteins by acting as ligands in affinity purification. In fusion expression vectors, proteolytic cleavage sites are often introduced at the junction of the fusion region and the recombinant protein to allow for the separation of the recombinant protein from the fusion region after purification of the fusion protein. Examples of such enzymes and their cognitive recognition sequences include factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A to the target recombinant protein, respectively.

[0044] Suitable examples of inducible non-fusion Escherichia coli (E. coli) expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0045] In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in budding yeast (Saccharomyces cerivisae) include pYepSec1 (Baldari, et al., 1987. EMBO J.6:229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30:933-943), pJRY88 (Schultz et al., 1987. Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[0046] In some embodiments, the vector uses a baculovirus expression vector to drive protein expression in insect cells. Examples of baculovirus vectors available for protein expression in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

[0047] In some embodiments, the vector may drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329:840) and pMT2PC (Kaufman, et al., 1987. EMBO J.6:187-195). When used in mammalian cells, the expression vector regulatory function is typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyomas, adenovirus type 2, cytomegalovirus, Simianvirus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0048] In some embodiments, recombinant mammalian expression vectors may preferentially direct the expression of nucleic acids in specific cell types (e.g., by using tissue-specific regulatory elements to express nucleic acids). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoters (liver-specific; Pinkert, et al., 1987. Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), particularly T cell receptor promoters (Winoto and Baltimore, 1989. EMBO J. 8:729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33:729-740; Queen and Baltimore, 1983. Cell 33:741-748), neuronal-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), and pancreas-specific promoters (Edlund, et al.) Examples include mammary gland-specific promoters (e.g., al., 1985. Science 230:912-916), and mammary gland-specific promoters (e.g., whey promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166). Developmental control promoters are also included, such as the mouse hox promoter (Kessel and Gruss, 1990. Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev.3:537-546).

[0049] In some embodiments, regulatory elements are operably bound to one or more elements of the CRISPR system to drive the expression of one or more elements of the CRISPR system. Generally, CRISPR (clustered equally spaced short repeats), also known as SPIDR (spacer interspersed direct repeats), constitute a family of DNA loci that are typically specific to certain bacterial species. CRISPR loci include distinct classes of interspersed short sequence repeats (SSRs) recognized in Escherichia coli (E. coli) (Ishino et al., J. Bacteriol., 169:5429-5433

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

[1989] ) and related genes. Similar scattered SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena species, and Mycobacterium tuberculosis (see Groenen et al., Mol. Microbiol., 10:1057-1065

[1993] ; Hoe et al., Emerg. Infect. Dis., 5:254-263

[1999] ; Masepohl et al., Biochim. Biophys. Acta 1307:26-30

[1996] ; and Mojica et al., Mol. Microbiol., 17:85-93

[1995] ). The CRISPR locus typically differs in repeat structure from other SSRs, and is referred to as a short-chain equispaced repeat (SRSR) (Janssen et al., OMICS J. Integ. Biol., 6:23-33

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

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

[2000] , op. cit.). Repeat sequences are highly conserved between strains,The number of scattered repeats and the sequence of spacer regions typically differ from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401

[2000] ). The CRISPR locus has been identified in more than 40 prokaryotes (e.g., Jansen et al., Mol. Microbiol., 43:1565-1575

[2002] ; and Mojica et al. (See al.,

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

[0050] Generally, the “CRISPR system” refers collectively to transcripts and other elements involved in the expression or orientation of the activity of CRISPR-related (“Cas”) genes, such as the sequence encoding the Cas gene, the tracr (trans-activated CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracrmate sequence (including “direct repeats” and partial direct repeats processed by tracrRNA with respect to the endogenous CRISPR system), the guide sequence (also referred to as “spacers” with respect to the endogenous CRISPR system), or other sequences and transcripts from the CRISPR locus. In some embodiments, one or more elements of the CRISPR system are derived from the type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from certain organisms containing the endogenous CRISPR system, such as Streptococcus pyogenes. Generally, the CRISPR system is characterized by elements that promote the formation of the CRISPR complex at a target sequence (also referred to as a protospacer in relation to the endogenous CRISPR system). With respect to the formation of the CRISPR complex, the “target sequence” refers to a sequence designed to have complementarity with a guide sequence, and hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Complete complementarity is not necessarily required, provided that sufficient complementarity exists to induce hybridization and promote the formation of the CRISPR complex. The target sequence may include any polynucleotide, e.g., DNA or RNA polynucleotide. In some embodiments, the target sequence is localized in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be located in an organelle of a eukaryotic cell, e.g., mitochondria or chloroplast. A sequence or template that can be used for recombination into a targeted locus containing the target sequence is referred to as an “editing template,” “edited polynucleotide,” or “editing sequence.” In embodiments of the present invention, an exogenous template polynucleotide may be referred to as an editing template. In one embodiment of the present invention, the recombination is homologous recombination.

[0051] Typically, with respect to the endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) results in the cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from it). Although not theoretically constrained, a tracr sequence may contain, or consist of, all or part of a wild-type tracr sequence (e.g., more than approximately 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), and may also form part of a CRISPR complex by hybridization along at least part of the tracr sequence with all or part of a tracr mate sequence operably bound to a guide sequence, for example. In some embodiments, the tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of the CRISPR complex. As with the target sequence, complete complementarity is not required, but sufficient complementarity may be present for it to be functional. In some embodiments, when optimally aligned, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence. In some embodiments, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into host cells, resulting in the expression of elements of the CRISPR system being directed towards the formation of the CRISPR complex at one or more target sites. For example, the Cas enzyme, a guide sequence bound to the tracr mate sequence, and the tracr sequence can each be operably bound to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector, and one or more additional vectors providing any components of the CRISPR system are not included in the first vector.CRISPR elements combined in a single vector can be positioned in any preferred orientation; for example, one element can be localized to the 5' side (upstream) or 3' side (downstream) of a second element. The coding sequence of one element can be localized on the same or reverse strand of the coding sequence of a second element and oriented in the same or reverse direction. In some embodiments, a single promoter drives the expression of a transcript encoding a CRISPR enzyme, as well as one or more guide sequences embedded within one or more intron sequences, tracr mate sequences (optionally operably bound to the guide sequence), and tracr sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably bound to the same promoter and expressed from there.

[0052] In some embodiments, the vector includes one or more insertion sites, e.g., restriction endonuclease recognition sequences (also referred to as “cloning sites”). In some embodiments, one or more insertion sites (e.g., about or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) are localized upstream and / or downstream of one or more sequence elements of one or more vectors. In some embodiments, the vector includes insertion sites upstream of a tracr-mate sequence and optionally downstream of a regulatory element operably bound to the tracr-mate sequence, so that after insertion of the guide sequence into the insertion site and upon expression, the guide sequence directs the CRISPR complex to sequence-specific binding to a target sequence in eukaryotic cells. In some embodiments, the vector includes two or more insertion sites, each insertion site localized between two tracr-mate sequences to allow insertion of the guide sequence at its respective site. In such arrangements, the two or more guide sequences may include two or more copies of a single guide sequence, two or more different guide sequences, or a combination thereof. When using multiple different guide sequences, a single expression construct can be used to target CRISPR activity against multiple different corresponding target sequences within a cell. For example, a single vector may contain approximately or more than approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, such guide sequence-containing vectors are provided and can optionally be delivered to cells.

[0053] In some embodiments, the vector includes a regulatory element operably bound to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-exclusive examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or modified versions thereof. These enzymes are publicly known; for example, the amino acid sequence of the *Streptococcus pyogenes* (S. pyogenes) Cas9 protein can be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity and is, for example, Cas9. In some embodiments, the CRISPR enzyme is Cas9 and may be Cas9 from *Streptococcus pyogenes* (S. pyogenes) or *Streptococcus pneumoniae* (S. pneumoniae). In some embodiments, the CRISPR enzyme is directed to cleave one or both strands within the target sequence and / or the complementary strand of the target sequence at the localization of the target sequence. In some embodiments, the CRISPR enzyme is directed to cleave one or both strands within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the vector encodes a CRISPR enzyme that is mutated from the corresponding wild-type enzyme, and as a result, the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence.For example, the substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes (S. pyogenes) (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that convert Cas9 to a nickase include, but are not limited to, H840A, N854A, and N863A. In some embodiments, the Cas9 nickase can be used in combination with guide sequences, e.g., two guide sequences that target the sense and antisense strands of the DNA target, respectively. This combination makes it possible to nick both strands and use them to induce an NHEJ. The applicants have demonstrated the efficacy of two nickase targets (i.e., sgRNAs that are identically localized but target different strands of DNA) in the induction of mutagenic NHEJs (data not shown). While a single nickase (Cas9-D10A with a single sgRNA) cannot induce NHEJ and create indels, we have shown that a dual nickase (Cas9-D10A and two sgRNAs targeted to different strands at the same localization) can do so in human embryonic stem cells (hESCs). The efficiency is approximately 50% of that of a nuclease (i.e., normal Cas9 without the D10 mutation) in hESCs.

[0054] As a further example, mutations in two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) can be used to produce mutant Cas9 that substantially lacks all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more H840A, N854A, or N863A mutations to produce a Cas9 enzyme that substantially lacks all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered substantially lacking all DNA cleavage activity if the DNA cleavage activity of the mutant enzyme is less than approximately 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less than that of its non-mutant form. Other mutations may be useful; if Cas9 or other CRISPR enzymes are from species other than Streptococcus pyogenes, mutations in the corresponding amino acids can be made to achieve similar effects.

[0055] In some embodiments, the enzyme coding sequence encoding a CRISPR enzyme is codon-optimized for expression in specific cells, eukaryotic cells, etc. Eukaryotic cells may be those of or derived from specific organisms, e.g., mammals, including, but not limited to, humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to improve expression in a target host cell by replacing at least one codon in the native sequence (e.g., more than approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more) with a more frequent or most frequent codon used in the host cell's genes, while maintaining the native amino acid sequence. Different species exhibit specific biases for certain codons of particular amino acids. Codon bias (differences in codon usage frequency between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which is then thought to depend, in particular, on the characteristics of the translated codon and the availability of specific transfer RNA (tRNA) molecules. The dominance of tRNAs selected in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Therefore, genes can be tuned for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in "codon usage databases," and these tables can be adapted using numerous methods. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon-optimizing specific sequences for expression in specific host cells are also available, for example, Gene Forge (Aptagen; Jacobus, PA).In some embodiments, one or more codons in the sequence encoding the CRISPR enzyme (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all of the codons) correspond to the codons most frequently used for a particular amino acid.

[0056] In some embodiments, the vector encodes a CRISPR enzyme containing one or more nuclear localization sequences (NLSs), e.g., about or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the CRISPR enzyme contains about or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino terminus, about or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy terminus, or a combination thereof (e.g., one or more NLSs at the amino terminus and one or more NLSs at the carboxy terminus). If two or more NLSs are present, each can be selected independently of the others, such that a single NLS may exist in two or more copies, and / or in combination with one or more other NLSs that exist in one or more copies. In a preferred embodiment of the present invention, the CRISPR enzyme contains at most six NLSs. In some embodiments, an NLS is considered to be located near the N or C terminus if its nearest neighbor amino acid is within approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N or C terminus. Typically, an NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the protein surface, although other types of NLSs are known.Non-limiting examples of NLS include: NLS of the SV40 virus large T antigen with the amino acid sequence PKKKRKV; NLS from nucleoplasmin (e.g., nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK); c-mycNLS with the amino acid sequence PAAKRVKLD or RQRRNELKRSP; hRNPA1 M9 NLS with the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; IBB domain sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from importin alpha; myoma T protein sequences VSRKRPRP and PPKKARED; human p53 sequence POPKKKPL; mouse c-abl Examples include the sequence SALIKKKKKMAP of influenza IV; the sequences DRLRR and PKQKKRK of influenza virus NS1; the sequence RKLKKKIKKL of hepatitis virus delta antigen; the sequence REKKKFLKRR of mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK of human poly(ADP-ribose) polymerase; and the NLS sequence derived from the sequence RKCLQAGMNLEARKTKK of the steroid hormone receptor (human) glucocorticoid.

[0057] Generally, one or more NLSs are strong enough to drive the accumulation of a detectable amount of CRISPR enzyme in the nucleus of eukaryotic cells. Generally, the strength of nuclear localization activity may depend on the number of NLSs in the CRISPR enzyme, the specific NLSs used, or a combination of these factors. Detection of nuclear accumulation can be carried out by any suitable technique. For example, a detectable marker can be fused to the CRISPR enzyme, and as a result, intracellular localization can be visualized in combination with means for detecting nuclear localization (e.g., nuclear-specific staining, e.g., DAPI). Examples of detectable markers include fluorescent proteins (e.g., green fluorescent protein, or GFP;RFP;CFP) and epitope tags (HA tags, flag tags, SNAP tags). Cell nuclei can also be isolated from cells, and their contents can then be analyzed by any suitable process for detecting proteins, e.g., immunohistochemical analysis, Western blotting, or enzyme activity assays. Nuclear accumulation can also be measured indirectly, for example, by assays for the effects of CRISPR complex formation (e.g., assays for DNA cleavage or mutation at target sequences, or assays for changes in gene expression activity affected by CRISPR complex formation and / or CRISPR enzyme activity), compared to controls that are not exposed to CRISPR enzymes or complexes, or are exposed to CRISPR enzymes lacking one or more NLSs.

[0058] Generally, the guide sequence is any polynucleotide sequence that hybridizes with the target sequence and has sufficient complementarity to the target polynucleotide sequence to direct the CRISPR complex to sequence-specific binding to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence exceeds approximately 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or greater, when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Examples include Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence has a nucleotide length of approximately or greater than approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more. In some embodiments, the guide sequence has a nucleotide length of approximately 75, 50, 45, 40, 35, 30, 25 The nucleotide lengths are 20, 15, 12, or less. The ability of a guide sequence to direct sequence-specific binding of the CRISPR complex to a target sequence can be evaluated by any suitable assay. For example, components of the CRISPR system sufficient to form a CRISPR complex, such as the guide sequence to be tested, can be provided to host cells having the corresponding target sequence, for example, by transtransfer using a vector encoding components of the CRISPR sequence, and then preferential cleavage within the target sequence can be evaluated by, for example, the Surveyor assay described herein.Similarly, cleavage of a target polynucleotide sequence can be evaluated by providing the target sequence, components of the CRISPR complex, e.g., a guide sequence to be tested and a control guide sequence different from the test guide sequence, in a test tube, and comparing the ratio of binding or cleavage at the target sequence between the test and control guide sequence reactions. Other assays are conceivable and will be recognized by those skilled in the art.

[0059] The guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the cell's genome. Exemplary target sequences include those unique within the target genome. For example, for Streptococcus pyogenes (S. pyogenes) Cas9, a unique target sequence in the genome could be the Cas9 target site of form MMMMMMMNNNNNNNNNNNNXGG, where NNNNNNNNNNNNXGG (where N is A, G, T, or C; X may be any) has a single occurrence in the genome. For S. thermophilus CRISPR1Cas9, the unique target sequence in the genome is the Cas9 target site of form MMMMMMMMNNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNXXAGAAW (where N is A, G, T, or C; X can be any; W is A or T) has a single development in the genome. The unique target sequence in the genome is the S. thermophilus CRISPR1Cas9 target site of form MMMMMMMMMNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNXXAGAAW (where N is A, G, T, or C; X can be any; W is A or T) has a single development in the genome. For Streptococcus pyogenes (S. pyogenes) Cas9, the unique target sequence in the genome is the Cas9 target site of form MMMMMMMNNNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXG (where N is A, G, T, or C; X may be any of these) has a single occurrence in the genome.A unique target sequence in the genome is the Streptococcus pyogenes (S. pyogenes) Cas9 target site of form MMMMMMMMMNNNNNNNNNNNXGGXG, where NNNNNNNNNNNXGGXG (N is A, G, T, or C; X may be any of these) has a single occurrence in the genome. In each of these sequences, "M" can be A, G, T, or C, and this does not need to be considered when identifying the sequence as unique.

[0060] In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of such an algorithm is mFold, described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is RNAfold, an online web server developed by the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (see, e.g., ARGruber et al., 2008, Cell 106(1):23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):1151-62). Further algorithms can be found in U.S. Patent Application No. TBA (Agent Reference No. 44790.11.2022; Broad Reference No. BI-2013 / 004A), which is incorporated herein by reference.

[0061] Generally, a tracr-mate sequence includes any sequence that has sufficient complementarity with the tracr sequence to facilitate one or more of the following: (1) the excision of a guide sequence flanked by the tracr-mate sequence in a cell containing the corresponding tracr sequence; and (2) the formation of a CRISPR complex at the target sequence (the CRISPR complex includes the tracr-mate sequence which hybridizes to the tracr sequence). Generally, the degree of complementarity is based on the optimal alignment of the tracr-mate sequence and the tracr sequence along the shorter of the two sequences. The optimal alignment can be determined by any preferred alignment algorithm, which may further describe secondary structures, such as self-complementarity within the tracr sequence or tracr-mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and the tracr-mate sequence along the shorter of the two sequences, when optimally aligned, exceeds approximately 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or greater. Figures 12B and 13B provide an illustrative description of the optimal alignment between the tracr sequence and the tracr mate sequence. In some embodiments, the tracr sequence has a nucleotide length of approximately or greater than approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more. In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript, resulting in hybridization between the two to produce a secondary structure, e.g., a transcript having a hairpin. A preferred loop-forming sequence used in a hairpin structure is 4 nucleotides long and most preferably has the sequence GAAA. However, longer or shorter loop sequences can be used, which may be alternative sequences. The sequence preferably comprises a nucleotide triplet (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In one embodiment of the present invention, the transcript or the polynucleotide sequence to be transcribed has at least two or more hairpins.In a preferred embodiment, the transcript has two, three, four, or five hairpins. In another further embodiment of the present invention, the transcript has at most five hairpins. In some embodiments, the single transcript further comprises a transcription termination sequence; preferably, this is a poly-T sequence, e.g., six T nucleotides. An exemplary description of such a hairpin structure is provided below in Figure 13B, where the last "N" and the sequence portion on the 5' side upstream of the loop correspond to the tracr mate sequence, and the sequence portion on the 3' side of the loop correspond to the tracr sequence. Further non-limiting examples of a single polynucleotide comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed from 5' to 3'), where "N" represents the base of the guide sequence, the first lowercase block represents the tracr mate sequence, the second lowercase block represents the tracr sequence, and the last poly-T sequence represents the transcription terminator. [ka] In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from the transcript containing the tracr mate sequence (for example, as illustrated in the upper figure of Figure 13B).

[0062] In some embodiments, recombinant templates are also provided. Recombinant templates are components of other vectors described herein and may be contained within a separate vector or provided as separate polynucleotides. In some embodiments, recombinant templates are designed to function as templates in or near a target sequence that are nicked or cleaved by a CRISPR enzyme as part of a CRISPR complex, for example. The template polynucleotide may be of any preferred length, e.g., a nucleotide length exceeding approximately or about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or a number greater than that. In some embodiments, the template polynucleotide is complementary to a portion of the polynucleotide containing the target sequence. When optimally aligned, the template polynucleotide may overlap with one or more nucleotides of the target sequence (e.g., more than approximately or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or a number greater than that). In some embodiments, when the polynucleotides containing the template sequence and the target sequence are optimally aligned, the nearest neighbor nucleotide of the template polynucleotide lies within approximately 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.

[0063] In some embodiments, the CRISPR enzyme is part of a fusion protein containing one or more heterologous protein domains (e.g., about one or more domains other than the CRISPR enzyme, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). The CRISPR enzyme fusion protein may contain any additional protein sequences and, optionally, linker sequences between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-exclusive 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, such as blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, such as, but are not limited to, maltose-binding protein (MBP), S-tags, Lex A DNA-binding domain (DBD) fusions, GAL4 DNA-binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein containing a CRISPR enzyme are described in U.S. Patent Application Publication No. 20110059502, which is incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the localization of a target sequence.

[0064] In some embodiments, the present invention provides methods for delivering one or more polynucleotides, e.g., or one or more vectors described herein, one or more transcripts thereof, and / or one or a protein transcribed therefrom, to a host cell. In some embodiments, the present invention further provides cells produced by such cells, and organisms (e.g., animals, plants, or fungi) comprising or produced therefrom such cells. In some embodiments, a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to the cell. Nucleic acids can be introduced into mammalian cells or target tissues using conventional viral and nonviral-based gene transfer methods. Using such methods, nucleic acids encoding components of the CRISPR system can be administered to cells in culture or into a host organism. Nonviral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and delivery vehicles, e.g., nucleic acids complexed with liposomes. Viral vector delivery systems include DNA and RNA viruses having genomes that are episomal or integrated after delivery to cells.For an overview of the gene therapy procedure, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and See Immunology, Doerfler and Boehm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0065] Nonviral delivery methods for nucleic acids include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced DNA uptake. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Suitable cations and neutral lipids for efficient receptor-recognized lipofection of polynucleotides include those from Felgner, International Publication No. 91 / 17424; International Publication No. 91 / 16024. Delivery may be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

[0066] The preparation of lipid-nucleic acid complexes, such as targeted liposomes, e.g., immunolipid complexes, is well known to those skilled in the art (e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820(1992); see U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0067] The use of RNA or DNA virus-based systems for nucleic acid delivery utilizes highly evolved processes to target viruses to specific cells in the body and transport the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo), or they can be used to treat cells in vitro, and in some cases, modified cells can be administered to patients (ex vivo). Conventional virus-based systems include retroviral, lentiviral, adenovirus, adeno-associated, and herpes simplex virus vectors for gene transfer. Integration into the host genome is considered for retroviral, lentiviral, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiencies have been observed in many different cell types and target tissues.

[0068] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, thereby expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that transduce or infect non-dividing cells and can typically produce high viral titers. Therefore, the selection of retroviral gene transfer systems depends on the target tissue. Retroviral vectors contain cis-acting long-chain terminal repeats (LTRs) that have the ability to package foreign sequences up to 6–10 kb. The minimum cis-acting LTR is sufficient for vector replication and packaging, which is then used to integrate therapeutic genes into target cells to provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), or combinations thereof (see, for example, Buchscher et al., J.Virol.66:2731-2739 (1992); Johann et al., J.Virol.66:1635-1640 (1992); Sommnerfelt et al., Virol.176:58-59 (1990); Wilson et al., J.Virol.63:2374-2378 (1989); Miller et al., J.Virol.65:2220-2224 (1991); and PCT / US94 / 05700). Adenovirus-based systems can be used in applications where transient expression is preferred. Adenovirus-based vectors can exhibit extremely high transduction efficiency in many cell types and do not require cell division. High titers and expression levels have been achieved with such vectors. These vectors can be produced in large quantities using relatively simple systems.For example, adeno-associated virus ("AAV") vectors can be used to transduce target nucleic acids into cells in the in vitro production of nucleic acids and peptides, as well as for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; International Publication No. 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors is described in numerous publications, e.g., U.S. Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al. This is described in al., Mol.Cell.Biol.4:2072-2081(1984); Hermonat & Muzyczka, PNAS 81:6466-6470(1984); and Samulski et al., J.Virol.63:03822-3828(1989).

[0069] Typically, packaging cells are used to form viral particles that can infect host cells. Examples of such cells include 293 cells for packaging adenoviruses, and ψ2 or PA317 cells for packaging retroviruses. Viral vectors used in gene therapy are usually produced by creating a cell system that packages nucleic acid vectors into viral particles. The vector typically contains the minimal viral sequence required for packaging and subsequent integration into the host, with other viral sequences replaced by expression cassettes for polynucleotides to be expressed. Deficient viral function is typically supplied trans by the packaging cell system. For example, AAV vectors used in gene therapy typically contain only the ITR sequence from the AAV genome required for packaging and integration into the host genome. The viral DNA is packaged within a cell system containing helper plasmids that encode other AAV genes, namely rep and cap, but lack the ITR sequence. The cell system can also be infected with adenovirus as a helper. Helper viruses facilitate the replication of AAV vectors and the expression of AAV genes from helper plasmids. Helper plasmids are not packaged in significant quantities due to the lack of ITR sequences. Adenovirus contamination can be reduced, for example, by heat treatment, in which adenoviruses are more susceptible than AAV. Additional methods for delivering nucleic acids to cells are known to those skilled in the art; see, for example, U.S. Patent Application Publication No. 20030087817, incorporated herein by reference.

[0070] In some embodiments, host cells are transfused transiently or nontransiently with one or more vectors described herein. In some embodiments, cells are transfused in their naturally occurring state within the subject. In some embodiments, the cells to be transfused are collected from the subject. In some embodiments, the cells are derived from cells collected from the subject, e.g., a cell line. A wide range of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, BALB / 3T3 mouse embryonic fibroblasts, 3T3Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis , A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR29 3, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells K562 cells, Ku8 12, KCL22, KG1, KYO1, LNCap, Ma-Mel1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK Examples include II, MOR / 0.2R, MONO-MAC6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lineage, Peer, PNT-1A / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lineage, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and their transgenic variants. Cell lines are available from various resources known to those skilled in the art (see, for example, the American Type Culture Collection (ATCC) (Manassus, Va.)). In some embodiments, a novel cell line containing one or more vector-derived sequences is established using cells transfused with one or more vectors described herein. In some embodiments, a novel cell line containing the modification but lacking any other exogenous sequences is established using cells transiently transfused with components of the CRISPR system described herein (e.g., transient transfusion with one or more vectors, or transfusion with RNA) and modified through the activity of the CRISPR complex. In some embodiments, cells transiently or nontransiently transfused with one or more vectors described herein, or cell lines derived from such cells, are used in the evaluation of one or more test compounds.

[0071] In some embodiments, one or more vectors described herein are used to produce non-human transgenic animals or transgenic plants. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, or rabbit. In some embodiments, the organism or subject is a plant. In some embodiments, the organism or subject or plant is an alga. Methods for producing transgenic plants and animals are known in the art and generally start from, for example, the cell translocation methods described herein.

[0072] In one embodiment, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises binding a CRISPR complex to the target polynucleotide to cause cleavage of the target polynucleotide, thereby modifying the target polynucleotide, the CRISPR complex comprising a CRISPR enzyme complexing with a guide sequence which hybridizes to a target sequence in the target polynucleotide, the guide sequence being bound to a tracr mate sequence which then hybridizes to a tracr sequence.

[0073] In one embodiment, the present invention provides a method for modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises conjugating a CRISPR complex to a polynucleotide such that the conjugation results in an increase or decrease in the expression of the polynucleotide; the CRISPR complex comprises a CRISPR enzyme complexing with a guide sequence which hybridizes to a target sequence in the target polynucleotide, the guide sequence being conjugated to a tracr mate sequence which then hybridizes to a tracr sequence.

[0074] With recent advances in crop genomics, the ability to perform efficient and cost-effective gene editing and manipulation using CRISPR-Cas systems enables the rapid selection and comparison of single and multiplex gene manipulations for transforming such genomes to improve production and enhance traits. In this regard, U.S. patents and publications: U.S. Patent No. 6,603,061 - Agrobacterium-Mediated Plant Transformation Method; U.S. Patent No. 7,868,149 - Plant Genome Sequences and Uses Thereof and U.S. Patent Application Publication No. 2009 / 0100536 - Transgenic Plants with Enhanced Agronomic Traits are referenced, and all of the contents and disclosures of each of these are incorporated herein by reference in their entirety. In the practice of the present invention, the contents and disclosures of Morrell et al. “Crop genomics: advances and applications” Nat Rev Genet. 2011 Dec 29;13(2):85-96 are also incorporated herein by reference in their entirety. In an advantageous embodiment of the present invention, microalgae are engineered using a CRISPR / Cas9 system (Example 15). Accordingly, references to animal cells herein may also apply to plant cells with necessary modifications, unless otherwise specified.

[0075] In one embodiment, the present invention provides a method for modifying a target polynucleotide in eukaryotic cells, which may be in vivo, ex vivo, or in vitro. In some embodiments, the method involves sampling cells or populations of cells from human or non-human animals or plants (including microalgae) and modifying one or more cells. Culturing can be performed ex vivo at any stage. One or more cells may also be reintroduced into non-human animals or plants (including microalgae).

[0076] In plants, pathogens are often host-specific. For example, Fusarium oxysporum f.sp. lycopersici causes tomato wilt but attacks only tomatoes, while Fusarium oxysporum f. dianthii and Puccinia graminis f.sp. tritici attack only wheat. Plants have pre-existing and inducible defenses to resist most pathogens. Mutation and recombination events throughout plant development result in genetic mutations that create susceptibility, especially when pathogens reproduce more frequently than plants. Non-host resistance can exist in plants, for example, when the host and pathogen are incompatible. Horizontal resistance, such as partial resistance to all species of a pathogen and vertical resistance, typically controlled by many genes, and complete resistance to some species of a pathogen but not others, typically controlled by a small number of genes, can also exist. At the gene-to-gene level, plants and pathogens evolve together, with genetic changes in one maintaining a balance with changes in others. Therefore, using natural variation, breeders combine genes most useful for yield, quality, uniformity, cold tolerance, and resistance. Resources for resistance genes include natural or introduced species, native species, wild plant relatives, and induced mutations, such as treating plant material with mutagenic agents. The present invention provides plant breeders with a novel tool for inducing mutations. Thus, those skilled in the art can analyze the genomes of resistance gene resources and use the present invention to induce the emergence of resistance genes in varieties with desired features or traits more accurately than conventional mutagenic agents, thereby accelerating and improving plant breeding programs.

[0077] In one embodiment, the present invention provides a kit containing one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit includes a vector system and instructions for use of the kit. In some embodiments, the vector system includes (a) a first regulatory element operably bound to one or more insertion sites for inserting a tracr mate sequence and a guide sequence upstream of the tracr mate sequence (the guide sequence, when expressed, directs the sequence-specific binding of the CRISPR complex to a target sequence in a eukaryotic cell, and the CRISPR complex includes a CRISPR enzyme that complexes with (1) a guide sequence hybridized to the target sequence and (2) a tracr mate sequence hybridized to the tracr sequence); and / or (b) a second regulatory element operably bound to an enzyme coding sequence encoding the CRISPR enzyme, including a nuclear localization sequence. The elements may be provided individually or in combination and may be provided in any suitable container, e.g., vials, bottles, or tubes. In some embodiments, the kit includes instructions in one or more languages, e.g., two or more languages.

[0078] In some embodiments, the kit includes one or more reagents used in a process utilizing one or more of the elements described herein. The reagents may be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. The reagents may be provided in a form usable in a particular assay, or in a form requiring the addition of one or more other components before use (e.g., in concentrate or lyophilized form). The buffer may be any buffer, including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10. In some embodiments, the kit includes one or more oligonucleotides corresponding to the guide sequence and the guide sequence for insertion into a vector for operably binding the regulatory element. In some embodiments, the kit includes homologous recombination template polynucleotides.

[0079] In one embodiment, the present invention provides a method using one or more elements of the CRISPR system. The CRISPR complex of the present invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the present invention has broad utility, for example, modification of a target polynucleotide (e.g., deletion, insertion, translocation, inactivation, activation) in a very large number of cell types. Therefore, the CRISPR complex of the present invention has a wide range of applications, for example, in gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary CRISPR complex comprises a CRISPR enzyme complexing with a guide sequence which hybridizes to a target sequence in the target polynucleotide. The guide sequence is then bound to a tracr-mate sequence which hybridizes to a tract sequence.

[0080] The target polynucleotide of the CRISPR complex can be any polynucleotide that is endogenous or exogenous to eukaryotic cells. For example, the target polynucleotide may be a polynucleotide that remains in the nucleus of a eukaryotic cell. The target polynucleotide may be a sequence that codes for a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). Although not constrained by theory, it is conceivable that the target sequence should associate with a PAM (protospacer adjacency motif); i.e., a short sequence recognized by the CRISPR complex. The exact sequence and length requirements for the PAM vary depending on the CRISPR enzyme used, but the PAM is typically a 2-5 base pair sequence adjacent to the protospacer (i.e., the target sequence). Examples of PAM sequences are given in the Examples section below, and those skilled in the art can identify further PAM sequences used for a given CRISPR enzyme.

[0081] Examples of target polynucleotides for the CRISPR complex include numerous disease-related genes and polynucleotides, as well as signaling biochemical pathway-related genes and polynucleotides, as listed in U.S. Provisional Patent Applications No. 61 / 736,527 and No. 61 / 748,427 (both titled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on December 12, 2012 and January 2, 2013, respectively, with Broad reference numbers BI-2011 / 008 / WSGR 44063-701.101 and BI-2011 / 008 / WSGR 44063-701.102, respectively; all of the contents of these applications are incorporated herein by reference as a whole).

[0082] Examples of targeted polynucleotides include sequences related to signaling biochemical pathways, e.g., signaling biochemical pathway-related genes or polynucleotides. Examples of targeted polynucleotides include disease-related genes or polynucleotides. A “disease-related” gene or polynucleotide refers to any gene or polynucleotide that produces a transcription or translation product at abnormal levels or in abnormal forms in cells derived from affected tissue compared to non-disease control tissue or cells. This may be a gene that becomes expressed at abnormally high levels; or it may be a gene that becomes expressed at abnormally low levels, and the change in expression correlates with the onset and / or progression of the disease. Disease-related genes also refer to genes that directly carry the pathogenesis of the disease, or genes that have mutations or genetic variations that are in linkage disequilibrium with genes that carry the pathogenesis of the disease. The transcription or translation product may be known or unknown, and may be at normal or abnormal levels.

[0083] Examples of disease-related genes and polynucleotides are available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), and are also available on the World Wide Web.

[0084] Examples of disease-related genes and polynucleotides are listed in Tables A and B. Disease-specific information is available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), and is also available on the World Wide Web. Examples of signaling biochemical pathway-related genes and polynucleotides are listed in Table C.

[0085] Mutations in these genes and pathways can result in the production of inappropriate proteins or inappropriate amounts of proteins affecting their function. Further examples of genes, diseases, and proteins are incorporated herein by reference from U.S. Provisional Patent Application No. 61 / 736,527, filed December 12, 2012, and U.S. Provisional Patent Application No. 61 / 748,427, filed February 2, 2013. Such genes, proteins, and pathways may be target polynucleotides of the CRISPR complex.

[0086] [Table 1]

[0087] [Table 2]

[0088] [Table 3]

[0089] [Table 4]

[0090] [Table 5]

[0091] Table 6

[0092] Table 7

[0093] Table 8

[0094] Table 9

[0095] Table 10

[0096] Table 11

[0097] Table 12

[0098] Table 13

[0099] Table 14

[0100] Table 15

[0101] [Table 16]

[0102] [Table 17]

[0103] Embodiments of the present invention also relate to methods and compositions related to gene knockout, gene amplification, and repair of specific mutations associated with DNA repeat instability and neurological diseases (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities and Neurological Diseases, Second Edition, Academic Press, Oct 13, 2011-Medical). Tandem repeat sequences of a defined form have been found to be responsible for more than 20 human diseases (New insights into repeat instability: role of RNA·DNA hybrids. McIvor EI, Polak U, Napierala M. RNA Biol. 2010 Sep-Oct;7(5):551-8). These abnormalities of genomic instability can be corrected using the CRISPR-Cas system.

[0104] A further aspect of the present invention relates to the use of a CRISPR-Cas system for correcting abnormalities in the EMP2A and EMP2B genes, which have been identified as being associated with Lafora disease. Olla disease is an autosomal recessive condition characterized by progressive myoclonic epilepsy, which can begin as epileptic seizures in adolescence. Several cases of this disorder may be caused by mutations in genes that have not yet been identified. The disorder progresses through seizures, muscle spasms, difficulty walking, dementia, and ultimately death. Currently, there is no treatment proven effective against disease progression. Other epilepsy-related genetic abnormalities can also be targeted using the CRISPR-Cas system, and the underlying genetics are further described in *Genetics of Epilepsy and Genetic Epilepsies*, edited by Giuliano Avanzini and Jeffrey L. Noebels, Mariani Foundation Paediatric Neurology: 20; 2009).

[0105] In yet another embodiment of the present invention, the CRISPR-Cas system is used in Genetic Diseases of the Eye, Second Edition, Elia It is possible to correct eye abnormalities resulting from several gene mutations, as further described in s I. Traboulsi, Oxford University Press, 2012.

[0106] Some further aspects of the present invention relate to the correction of abnormalities associated with a wide range of genetic disorders, which are further described on the website of the National Institutes of Health (health.nih.gov / topic / GeneticDisorders) under the topic subsection Genetic Disorders. Genetic brain disorders include, but are not limited to, adrenoleukodystrophy, corpus callosum agenesis, Aicardi syndrome, Alpers disease, Alzheimer's disease, Barth syndrome, Batten disease, CADASIL, cerebellar degeneration, Fabry disease, Gerstmann-Streussler-Scheinker disease, Huntington's disease and other triplet repeat diseases, Leigh disease, Lesch-Nyhan syndrome, Menkes disease, mitochondrial myopathy, and NINDS colposephrosis. These disorders are further described on the website of the National Institutes of Health under the subsection Genetic Brain Disorders.

[0107] In some embodiments, the pathophysiology is neoplastic. In some embodiments where the pathophysiology may be neoplastic, the gene to be targeted may be one of those listed in Table A (in this case, PTEN, etc.). In some embodiments, the pathophysiology may be age-related macular degeneration. In some embodiments, the pathophysiology may be schizophrenia. In some embodiments, the pathophysiology may be trinucleotide repeat disorder. In some embodiments, the pathophysiology may be fragile X syndrome. In some embodiments, the pathophysiology may be secretase-related disorder. In some embodiments, the pathophysiology may be prion-related disorder. In some embodiments, the pathophysiology may be ALS. In some embodiments, the pathophysiology may be drug addiction. In some embodiments, the pathophysiology may be autism. In some embodiments, the pathophysiology may be Alzheimer's disease. In some embodiments, the pathophysiology may be inflammation. In some embodiments, the pathophysiology may be Parkinson's disease.

[0108] Examples of proteins associated with Parkinson's disease, though not limited to them, include α-synuclein, DJ-1, LRRK2, PINK1, parkin, UCHL1, synphyrin-1, and NURR1.

[0109] An example of a preference-related protein is ABAT.

[0110] Examples of inflammation-related proteins include monocyte chemotactic protein-1 (MCP1) encoded by the Ccr2 gene, CC chemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, IgG receptor IIB (FCGR2b, also known as CD32) encoded by the Fcgr2b gene, or Fc epsilon R1g (FCER1g) protein encoded by the Fcer1g gene.

[0111] Examples of cardiovascular disease-related proteins include IL1B (interleukin-1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin I2 (prostacyclin) synthase), MB (myoglobin), IL4 (interleukin-4), ANGPT1 (angiopoietin-1), ABCG8 (ATP-binding cassette, subfamily G (WHITE), member 8), or CTSK (cathepsin K).

[0112] Examples of Alzheimer's disease-related proteins include, for example, the very low-density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, ubiquitin-like modifier activator enzyme 1 (UBA1) encoded by the UBA1 gene, or the NEDD8 activator enzyme E1 catalytic subunit protein (UBE1C) encoded by the UBA3 gene.

[0113] Examples of proteins associated with autism spectrum disorder include, for example, benzodiazepine receptor (peripheral)-associated protein 1 (BZRAP1) encoded by the BZRAP1 gene, AF4 / FMR2 family member 2 protein (AFF2) encoded by the AFF2 gene (also known as MFR2), fragile X autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene, or fragile X autosomal homolog 2 protein (FXR2) encoded by the FXR2 gene.

[0114] Examples of proteins associated with macular degeneration include, for example, ATP-binding cassette subfamily A (ABC1) member 4 protein (ABCA4) encoded by the ABCR gene, apolipoprotein E protein (APOE) encoded by the APOE gene, or chemokine (CC motif) ligand 2 protein (CCL2) encoded by the CCL2 gene.

[0115] Examples of proteins associated with schizophrenia include NRG1, ErbB4, CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, and combinations thereof.

[0116] Examples of proteins involved in tumor suppression include ATM (ataxia telangiectasia mutation), ATR (ataxia telangiectasia and Rad3-related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia virus oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia virus oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia virus oncogene homolog 4), Notch1, Notch2, Notch3, or Notch4.

[0117] Examples of proteins associated with secretase disorders include PSENEN (presenilin enhancer 2 homolog (nematode (C. elegans))), CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid beta (A4) precursor protein), APH1B (prepharyngeal deficiency 1 homolog B (nematode (C. elegans))), PSEN2 (presenilin 2 (Alzheimer's disease 4)), or BACE1 (beta-site APP cleavage enzyme 1).

[0118] Examples of proteins associated with amyotrophic lateral sclerosis (ALS) include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA-binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), as well as any combination thereof.

[0119] Examples of proteins associated with prion diseases include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA-binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof.

[0120] Examples of proteins associated with neurodegenerative pathologies in prion disorders include, for example, A2M (alpha-2 macroglobulin), AATF (anti-apoptotic transcription factor), ACPP (prostatic acid phosphatase), ACTA2 (aortic smooth muscle actin alpha-2), ADAM22 (ADAM metallopeptidase domain), ADORA3 (adenosine A3 receptor), or ADRA1D (alpha-1D adrenergic receptor for alpha-1D adrenergic receptor).

[0121] Examples of proteins associated with immunodeficiency include, for example, A2M [alpha-2-macroglobulin]; AANAT [arylalkylamine N-acetyltransferase]; ABCA1 [ATP-binding cassette subfamily A (ABC1), member 1]; ABCA2 [ATP-binding cassette subfamily A (ABC1), member 2]; or ABCA3 [ATP-binding cassette subfamily A (ABC1), member 3].

[0122] Examples of proteins associated with trinucleotide repeat disorders include, for example, AR (androgen receptor), FMR1 (fragility x intellectual disability 1), HTT (Huntington's syndrome), and These include DMPK (myotonic dysplasia protein kinase), FXN (frataxin), and ATX N2 (ataxin 2) is one example.

[0123] Examples of proteins associated with neurotransmission disorders include, for example, SST (somatostatin), NOS1 (nitric oxide synthase 1 (neuronal type)), ADRA2A (adrenergic alpha-2A receptor), ADRA2C (adrenergic alpha-2C receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-hydroxytryptamine (serotonin) receptor 2C).

[0124] Examples of neurodevelopment-related sequences include, for example, A2BP1 [ataxin 2-binding protein 1], AADAT [aminoadipate aminotransferase], AANAT [arylalkylamine N-acetyltransferase], ABAT [4-aminobutyrate aminotransferase], ABCA1 [ATP-binding cassette subfamily A (ABC1) member 1], or ABCA13 [ATP-binding cassette subfamily A (ABC1) member 13].

[0125] Further examples of preferred conditions treatable by the system of the present invention can be selected from the following: Alcardi-Gutierre syndrome; Alexander disease; Alan-Herndon-Dudley syndrome; POLG-related disorder; alpha-mannosidosis (types II and III); Alström syndrome; Angelman syndrome; ataxia with telangiectasia; neuronal ceroid lipofuscinosis; beta-cerasamia; bilateral optic atrophy and (infantile) optic atrophy type 1; retinoblastoma (bilateral); Canavan disease; cerebro-ocular-facial-skeletal syndrome 1 [COFS1]; cerebral tendon xanthomas Syndrome; Cornelia de Lang syndrome; MAPT-related disorders; hereditary prion diseases; Dravet syndrome; early-onset familial Alzheimer's disease; Friedreich's ataxia [FRDA]; Flins syndrome; fucosidosis; Fukuyama-type congenital muscular dystrophy; galactosialidosis; Gaucher disease; organic acidemia; hemophagocytic lymphohistiocytosis; Hutchinson-Gilford progeria syndrome; mucolipidosis type II; infantile free sialic acid storage; PLA2G6-related neurodegeneration; Javer-Lange-Nielsen syndrome; junctional epidermolysis bullosa; Huntington's disease; Krabbe disease (infant type) Mitochondrial DNA-related Lie syndrome and NARP; Lesch-Nyhan syndrome; LIS1-related lissencephaly; Lowe syndrome; Maple syrup urine disease; MECP2 duplication syndrome; ATP7A-related copper transport disorder; LAMA2-related muscular dystrophy; Arylsulfatase A deficiency; Mucopolysaccharidosis type I, II, or III; Peroxisome dysplasia, Zellweger syndrome spectrum; Neurodegenerative diseases with cerebral iron storage; Acid sphingomyelinase deficiency; Niemann-Pick disease type C; Glycine encephalopathy; ARX-related disorders; Urea cycle disorders; COL1A1 / 2-related disorders Osteogenesis imperfecta; Mitochondrial DNA deletion syndrome; PLP1-related disorder; Perry syndrome; Phelan-McDermott syndrome; Glycogen storage disorder type II (Pompe disease) (infant form); MAPT-related disorder; MECP2-related disorder; Root chondrodysplasia type 1; Roberts syndrome; Sandhoff disease; Schindler disease type 1; Adenosine deaminase deficiency; Smith-Lemley-Opitz syndrome; Spinal muscular atrophy; Infant-onset spinocerebellar ataxia; Hexosaminidase A deficiency; Fatal dysplasia type 1; Collagen type VI-related disorder; Usher syndrome type I; Congenital muscular dystrophy;Wolff-Hirschhorn syndrome; lysosomal acid lipase deficiency; and xeroderma pigmentosum.

[0126] As is evident, it is assumed that any target polynucleotide sequence can be targeted using the system of the present invention. Some examples of conditions or diseases that can be usefully treated using the system of the present invention are included in the table above, and examples of genes currently associated with those conditions are also provided in the table. However, the exemplified genes are not exclusive. [Examples]

[0127] The following examples are given for the purpose of illustrating various embodiments of the present invention, and are not intended to limit the invention to any particular form. These examples, along with the methods described herein, are representative and illustrative examples of currently preferred embodiments and are not intended to limit the scope of the invention. Modifications and other uses incorporated therein into the spirit of the invention as defined by the claims will be made by those skilled in the art.

[0128] Example 1: CRISPR complex activity in the nucleus of eukaryotic cells An exemplary type II CRISPR system is a type II CRISPR locus from Streptococcus pyogenes SF370 containing a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as a characteristic array of repetitive sequences (direct repeats) spaced by two non-coding RNA elements, tracrRNA, and short stretches of non-repetitive sequences (spacers, each approximately 30 bp). In this system, targeted DNA double-strand breaks (DSBs) are generated in four sequential steps (Figure 2A). First, two non-coding RNAs, a pre-crRNA array, and tracrRNA are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to the direct repeats of the pre-crRNA, which is then processed into mature crRNA containing individual spacer sequences. Thirdly, the mature crRNA:tracrRNA complex directs Cas9 to a DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the crRNA spacer region and the protospacer DNA. Finally, Cas9 creates a double-strand break (DSB) within the protospacer by mediating the cleavage of the target DNA upstream of the PAM (Figure 2A). This example describes an exemplary process for adapting this RNA programmable nuclease system to direct CRISPR complex activity in the nucleus of a eukaryotic cell.

[0129] Cell culture and translocation Human embryonic kidney (HEK) cell line HEK293FT (Life Technologies) was maintained at 37°C under 5% CO2 incubation in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100 U / mL penicillin, and 100 μg / mL streptomycin. Mouse neuro2A (N2A) cell line (ATCC) was maintained at 37°C under 5% CO2 in DMEM supplemented with 5% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0130] HEK293FT or N2A cells were seeded in 24-well plates (Corning) at a density of 200,000 cells per well one day prior to translocation. Cells were translocated using Lipofectamine 2000 (Life Technologies) according to the manufacturer's recommended protocol. A total of 800 ng of plasmid was used for each well of the 24-well plate.

[0131] Surveyor assays and sequencing analyses of genome modifications HEK293FT or N2A cells were transfused with the plasmid DNA described above. After transfusion, the cells were incubated at 37°C for 72 hours, and then genomic DNA was extracted. Genomic DNA was extracted using the QuickExtract DNA extraction kit (Epicentre) according to the manufacturer's protocol. Briefly, the cells were resuspended in QuickExtract solution and incubated at 65°C for 15 minutes and at 98°C for 10 minutes. The extracted genomic DNA was processed immediately or stored at -20°C.

[0132] The genomic region surrounding the CRISPR target site for each gene was PCR-amplified, and the product was purified using a QiaQuick Spin Column (Qiagen) according to the manufacturer's protocol. A total of 400 ng of purified PCR product was mixed with 2 μl of 10× Taq polymerase PCR buffer (Enzymatics), and diluted with ultrapure water to a final volume of 20 μl. This was then subjected to a re-nealing process to enable heteroduplex formation: 95°C for 10 minutes, then 95°C to 85°C with a gradient of -2°C / sec, 85°C to 25°C with a gradient of -0.25°C / sec, and finally maintained at 25°C for 1 minute. After re-nealing, the product was treated with Surveyor nuclease and Surveyor enhancer S (Transgenomics) according to the manufacturer's recommended protocol and analyzed on 4–20% Novex TBE polyacrylamide gel (Life Technologies). The gel was stained with SYBR Gold DNA stain (Life Technologies) for 30 minutes and imaged using the Gel Doc gel imaging system (Bio-rad). Quantification was based on relative band intensity as a measure of the rate of cleaved DNA. Figure 8 provides a schematic explanation of this Surveyor assay.

[0133] Restriction fragment length polymorphism assay for the detection of homologous recombination HEK293FT and N2A cells were transfused with plasmid DNA, incubated at 37°C for 72 hours, and then genomic DNA was extracted as described above. Target genomic regions were PCR amplified using primers outside the homology arms of the homologous recombination (HR) template. PCR products were separated on a 1% agarose gel and extracted using the MinElute Gel Extraction Kit (Qiagen). The purified products were digested with HindIII (Fermentas) and analyzed on a 6% Novex TBE polyacrylamide gel (Life Technologies).

[0134] RNA secondary structure prediction and analysis RNA secondary structure prediction was performed using the RNAfold online web server developed at the Institute for Theoretical Chemistry at the University of Vienna, employing a centroid structure prediction algorithm (see, e.g., ARGruber et al., 2008, Cell 106(1):23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):1151-62).

[0135] Bacterial plasmid transformation interference assay Elements of the CRISPR locus 1 of Streptococcus pyogenes (S. pyogenes) sufficient for CRISPR activity were reconstituted in Escherichia coli (E. coli) using a pCRISPR plasmid (schematically illustrated in Figure 10A). The pCRISPR plasmid contained tracrRNA, SpCas9, and a leader sequence to drive the crRNA array. A spacer (also referred to as the "guide sequence") was inserted between BsaI sites in the crRNA array using annealed oligonucleotides as described. The challenge plasmid used in the interference assay was constructed by inserting a protospacer (also referred to as the "target sequence") sequence into pUC19 along with an adjacent CRISPR motif sequence (PAM) (see Figure 10B). The challenge plasmid contained ampicillin resistance. Figure 10C provides a schematic representation of the interference assay. Chemically competent Escherichia coli (E. coli) strains already carrying pCRISPR and appropriate spacers were transformed with challenge plasmids containing the corresponding protospacer-PAM sequence. The transformation efficiency of each pCRISPR-carrying competent strain was evaluated using pUC19. CRISPR activity resulted in cleavage of the protospacer-carrying pPSP plasmid, excluding ampicillin resistance otherwise conferred by pUC19 lacking the protospacer. Figure 10D illustrates the competence of each pCRISPR-carrying Escherichia coli (E. coli) strain used in the assay described in Figure 4C.

[0136] RNA purification HEK293FT cells were maintained and transfused as described above. The cells were harvested by trypsin treatment and then washed in phosphate-buffered saline (PBS). Total cellular RNA was extracted using TRI reagent (Sigma) according to the manufacturer's protocol. The extracted total RNA was quantified using Naonodrop (Thermo Scientific) and normalized to the same concentration.

[0137] Northern blot analysis of crRNA and tracrRNA expression in mammalian cells RNA was mixed with an equal volume of 2× loading buffer (Ambion), heated to 95°C for 5 minutes, refrigerated on ice for 1 minute, and then loaded onto an 8% denatured polyacrylamide gel (SequaGel, National Diagnostics) after at least 30 minutes of gel prerun. The samples were electrophoresed at a 40W limit for 1.5 hours. Subsequently, the RNA was transferred to a Hybond N+ membrane (GE Healthcare) at 300 mA in a semi-dry transfer device (Bio-rad) at room temperature for 1.5 hours. The RNA was crosslinked to the membrane using the auto-crosslink button on the Stratalinker (Stratagene) of a Stratagene UV Crosslinker. The membrane was pre-hybridized at 42°C for 30 minutes while rotating in ULTRAhyb-oligohybridization buffer (Ambion), then the probe was added, and hybridization was carried out overnight. The probes were ordered from IDT and labeled with [gamma-32P]ATP (Perkin Elmer) using T4 polynucleotide kinase (New England Biolabs). The membranes were preheated (42°C) and washed once for 1 minute with 2×SSC and 0.5% SDS, then washed twice for 30 minutes at 42°C. The membranes were exposed to a fluorescent screen at room temperature for 1 hour or overnight, and then scanned with a phosphorimager (Typhoon).

[0138] Construction and evaluation of bacterial CRISPR systems CRISPR locus elements, including tracrRNA, Cas9, and the leader, were PCR-amplified from Streptococcus pyogenes SF370 genomic DNA using flanking homology arms for Gibson assembly. Two BsaI IIS-type sites were introduced between two direct repeats to facilitate spacer insertion (Figure 9). PCR products were cloned downstream of the tet promoter into EcoRV digested pACYC184 using Gibson Assembly Master Mix (NEB). The last 50 bp of Csn2 were removed to exclude other endogenous CRISPR elements. An oligo encoding a spacer with a complementary overhang (Integrated DNA Technology) was cloned into the BsaI digested vector pDC000 (NEB) and then ligated with T7 ligase (Enzymatics) to generate a pCRISPR plasmid. A challenge plasmid containing a spacer having a PAM sequence (also referred to herein as a "CRISPR motif sequence") was created by ligating a hybridized oligo (Integrated DNA Technology) carrying an equivalent overhang into BamHI-digested pUC19. Cloning for all constructs was performed in Escherichia coli (E. coli) strain JM109 (Zymo Research).

[0139] pCRISPR-carrying cells were prepared using the Z-Competent E. coli Transformation Kit and Buffer Set (Zymo Research, T3001) according to the manufacturer's instructions. In the transformation assay, 50 μL aliquots of pCRISPR-carrying competent cells were thawed on ice, transformed with 1 ng of spacer plasmid or pUC19 on ice for 30 minutes, then heat-shocked at 42°C for 45 seconds and maintained on ice for 2 minutes. Subsequently, 250 μL of SOC (Invitrogen) was added, followed by incubation at 37°C for 1 hour with shaking. 100 μL of the post-SOC pre-culture was plated onto a dual-selection plate (12.5 μg / ml chloramphenicol, 100 μg / ml ampicillin). The total number of colonies was multiplied by 3 to obtain 1 ng of cfu / DNA.

[0140] To improve the expression of CRISPR components in mammalian cells, two genes from the Streptococcus pyogenes (S. pyogenes) SF370 locus 1, Cas9 (SpCas9) and RNase III (SpRNase III), were codon-optimized. To promote nuclear localization, a nuclear localization signal (NLS) was included at the amino (N) or carboxyl (C) terminus of both SpCas9 and SpRNase III (Figure 2B). To facilitate the visualization of protein expression, a fluorescent protein marker was also included at the N or C terminus of both proteins (Figure 2B). A version of SpCas9 with NLS attached to both the N and C termini (2×NLS-SpCas9) was also generated. Constructs containing NLS-fused SpCas9 and SpRNase III were transfused into 293FT human embryonic kidney (HEK) cells, and it was found that the relative positioning of NLS to SpCas9 and SpRNase III affected their nuclear localization efficiency. While C-terminal NLS was sufficient to target the target SpRNase III to the nucleus, attachment of a single copy of these specific NLS to either the N or C-terminus of SpCas9 failed to achieve adequate nuclear localization in this system. In this example, the C-terminal NLS was that of nucleoplasmin (KRPAATKKAGQAKKKK), and the C-terminal NLS was that of SV40 large T antigen (PKKKRKV). Of the versions of SpCas9 tested, only 2×NLS-SpCas9 showed nuclear localization (Figure 2B).

[0141] TracrRNA from the CRISPR locus of Streptococcus pyogenes SF370 has two transcription start sites, producing two transcripts of 89 nucleotides (nt) and 171 nt, which are subsequently processed into the same 75 nt mature tracrRNA. The shorter 89 nt tracrRNA was selected for expression in mammalian cells (with the expression construct described in Figure 7A and functionality determined by the Surveyor assay results shown in Figure 7B). The transcription start site is labeled as +1, and the transcription terminator and sequences probed by Northern blotting are also shown. Expression of the processed tracrRNA was also confirmed by Northern blotting. Figure 7C shows the results of Northern blotting analysis of total RNA extracted from 293FT cells transfected with long or short tracrRNAs, as well as U6 expression constructs carrying SpCas9 and DR-EMX1(1)-DR. The left and right panels are from 293FT cells transfused with or without SpRNase III, respectively. U6 shows the loading control blotted with a probe targeting human U6 snRNA. Transfusion with the short tracrRNA expression construct resulted in a sufficient level of processed tracrRNA (approximately 75 bp). Very small amounts of long tracrRNA were detected on the Northern blot.

[0142] To promote accurate transcription initiation, we selected an RNA polymerase III-based U6 promoter to drive tracrRNA expression (Figure 2C). Similarly, we developed a U6 promoter-based construct to express a precrRNA array consisting of a single spacer flanked by two direct repeats (DRs, also encompassed in the term "tracrmate sequence"; Figure 2C). The first spacer was designed to target a 33-base pair (bp) target site in the human EMX1 locus, a key gene in cerebral cortex development (a 30-bp protospacer and a 3-bp CRISPR motif (PAM) sequence that satisfies the Cas9 NGG recognition motif) (Figure 2C).

[0143] To investigate whether heterologous expression of the CRISPR system (SpCas9, SpRNase III, tracrRNA, and precrRNA) in mammalian cells could achieve targeted mammalian chromosome cleavage, HEK293FT cells were transfused with combinations of CRISPR components. Since double-segment breaks (DSBs) in mammalian nuclei are partially repaired by the non-homologous end joining (NHEJ) pathway, which leads to indel formation, potential cleavage activity at the target EMX1 locus was detected using a Surveyor assay (Figure 8) (see, e.g., Guschin et al., 2010, Methods Mol Biol 649:247). Simultaneous transfusion of all four CRISPR components was able to induce cleavage of up to 5.0% of protospacers (see Figure 2D). Simultaneous translocation of all CRISPR components except SpRNase III also induced up to 4.7% of indels in the protospacer, suggesting the presence of endogenous mammalian RNases, such as the associated Dicer and Drosha enzymes, that may support crRNA maturation. Removal of any of the remaining three components halted the genome-cleaving activity of the CRISPR system (Figure 2D). Sanger sequencing of amplicons containing the target locus confirmed the cleavage activity; five mutant alleles (11.6%) were found among 43 sequenced clones. Similar experiments using various guide sequences resulted in a higher indel rate of 29% (see Figures 4-7, 12, and 13). These results define a three-component system for efficient CRISPR-mediated genome modification in mammalian cells. To optimize cleavage efficiency, the applicants also tested whether different isoforms of tracrRNA affect cleavage efficiency and found that in this exemplary system, only the short-chain (89 bp) transcript morphology could mediate the cleavage of the human EMX1 genome locus (Figure 7B).

[0144] Figure 14 provides an additional Northern blot analysis of crRNA processing in mammalian cells. Figure 14A illustrates a schematic diagram showing the expression vector for a single spacer (DR-EMX1(1)-DR) flanked by two direct repeats. The 30 bp spacer (see Figure 6) and direct repeat sequences targeting human EMX1 locus protospacer 1 are shown in the lower sequence of Figure 14A. Lines indicate regions that generate a Northern blot probe for EMX1(1) crRNA detection using the reverse complementary sequence. Figure 14B shows a Northern blot analysis of total RNA extracted from 293FT cells transfected with a U6 expression construct carrying DR-EMX1(1)-DR. The left and right panels are from 293FT cells transfected with and without SpRNase III, respectively. DR-EMX1(1)-DR was processed into mature crRNA only in the presence of SpCas9, and short tracrRNA was independent of the presence of SpRNase III. The mature crRNA detected from transfected 293FT total RNA was approximately 33 bp, shorter than the 39–42 bp mature crRNA from Streptococcus pyogenes. These results demonstrate that the CRISPR system can be transplanted into eukaryotic cells and reprogrammed to promote the cleavage of endogenous mammalian target polynucleotides.

[0145] Figure 2 illustrates the bacterial CRISPR system described in this embodiment. Figure 2A illustrates CRISPR locus 1 from Streptococcus pyogenes SF370 and a schematic diagram showing the proposed mechanism of CRISPR-mediated DNA cleavage by this system. Mature crRNA processed from a direct repeat-spacer array directs Cas9 to a genomic target consisting of a complementary protospacer and a protospacer-adjacent motif (PAM). During target-spacer base pairing, Cas9 mediates double-strand breaks in the target DNA. Figure 2B illustrates the engineering of Streptococcus pyogenes Cas9 (SpCas9) and RNase III (SpRNase III) with nuclear localization signals (NLS) to enable transport into the mammalian nucleus. Figure 2C illustrates the mammalian expression of SpCas9 and SpRNase III driven by the constitutive EF1a promoter, as well as tracrRNA and precrRNA arrays (DR-spacer-DR) driven by the RNAPol3 promoter U6 to promote accurate transcription initiation and termination. A protospacer from the human EMX1 locus with sufficient PAM sequences is used as a spacer in the precrRNA array. Figure 2D illustrates a surveyor nuclease assay for SpCas9-mediated minor insertions and deletions. SpCas9 was expressed with or without the precrRNA array carrying SpRNase III, tracrRNA, and the EMX1-targeting spacer. Figure 2E illustrates a schematic representation of base pairing between the target locus and the EMX1-targeting crRNA, as well as an exemplary chromatogram showing a microdeletion adjacent to the SpCas9 cleavage site. Figure 2F illustrates mutant alleles identified from sequencing analysis of 43 clonal amplicons exhibiting various microinsertions and deletions. Dotted lines indicate deleted bases; unaligned or mismatched bases indicate insertions or mutations. Scale bar = 10 μm.

[0146] To further simplify the three-component system, a chimeric crRNA-tracrRNA hybrid design mimicking the natural crRNA:tracrRNA double-strand was adapted by fusing mature crRNA (including the guide sequence) to a partial tracrRNA via a stem-loop (Figure 3A). To increase simultaneous delivery efficiency, a bicistronic expression vector was created to drive the simultaneous expression of chimeric RNA and SpCas9 in translocation cells (Figures 3A and 38). In parallel, precrRNA (DR-guide sequence-DR) ​​was expressed together with SpCas9 using the bicistronic vector to induce processing to crRNA, and tracrRNA was expressed separately (compare the upper and lower figures in Figure 13B). Figure 9 provides a schematic description of a bicistronic expression vector for a pre-crRNA array with hSpCas9 (Figure 9A) or a chimeric crRNA (represented by short lines downstream of the guide sequence insertion site and upstream of the EF1α promoter in Figure 9B), showing the localization of various elements and the site of guide sequence insertion. The magnified sequence around the localization of the guide sequence insertion site in Figure 9B also shows the partial DR sequence (GTTTAGAGCTA) and the partial tracrRNA sequence (TAGCAAGTTAAAATAAGGCTAGTCCGTTTTT). The guide sequence can be inserted between the BbsI sites using annealed oligonucleotides. Sequence designs for oligonucleotides are shown below the schematic description in Figure 9, indicating appropriate ligation adapters. WPRE represents the woodchuck hepatitis virus post-transcriptional regulatory element. The efficiency of chimeric RNA-mediated cleavage was tested by targeting the same EMX1 locus described above. Using both the Surveyor assay and Sanger sequencing of amplicons, the applicants confirmed that chimeric RNA design promotes cleavage of the human EMX1 locus with a modification rate of approximately 4.7% (Figure 4).

[0147] The generalizability of CRISPR-mediated cleavage in eukaryotic cells was tested by targeting additional genomic loci in both human and mouse cells by designing chimeric RNAs that target multiple sites within the human EMX1 and PVALB loci, as well as the mouse Th locus. Figure 15 illustrates the selection of protospacers in several additional targetable human PVALB (Figure 15A) and mouse Th (Figure 15B) loci. It provides schematic diagrams of the loci and the localization of the three protospacers within their respective last exons. Underlined sequences include a 30 bp protospacer sequence and a 3 bp protospacer sequence at the 3' end corresponding to the PAM sequence. Protospacers on the sense and antisense strands are shown above and below the DNA sequence, respectively. Modification ratios of 6.3% and 0.75% were achieved for the human PVALB and mouse Th loci, respectively, demonstrating the broad applicability of the CRISPR system in modifying different loci across multiple organisms (Figures 3B and 6). While cleavage was detected in only one of the three spacers for each locus using a chimeric construct, all target sequences were cleaved with an indel generation efficiency of 27% when using a co-expressed precrRNA configuration (Figure 6).

[0148] Figure 13 provides further explanation of how SpCas9 can be reprogrammed to target multiple genomic loci in mammalian cells. Figure 13A provides a schematic diagram of the human EMX1 locus showing the localization of five protospacers indicated by underlined sequences. Figure 13B provides a schematic diagram of the precrRNA / trcrRNA complex (top) showing hybridization between direct repeat regions of precrRNA and tracrRNA, a 20 bp guide sequence, and a schematic diagram of a chimeric RNA design (bottom) including a tracrmate and tracr sequence consisting of a partial direct repeat and tracrRNA sequence hybridized into a hairpin structure. Figure 13C illustrates the results of a Surveyor assay comparing the efficacy of Cas9-mediated cleavage in the five protospacers in the human EMX1 locus. Each protospacer is targeted using either the processed precrRNA / tracrRNA complex (crRNA) or chimeric RNA (chiRNA).

[0149] Because RNA secondary structure can be important for intermolecular interactions, we compared the predicted secondary structures of all guide sequences used in our genome targeting experiments using a structure prediction algorithm based on minimum free energy and Boltzmann weighted structure ensemble (Figure 3B) (see, e.g., Gruber et al., 2008, Nucleic Acids Research, 36:W70). The analysis revealed that, in most cases, effective guide sequences in the chimeric crRNA context substantially lack secondary structure motifs, while ineffective guide sequences are more likely to form internal secondary structures that can interfere with base pairing with target protospacer DNA. Therefore, variability in spacer secondary structure may affect the efficiency of CRISPR-mediated interference when using chimeric crRNA.

[0150] Figure 3 illustrates exemplary expression vectors. Figure 3A provides a schematic diagram of a bicistronic vector for driving the expression of a synthetic crRNA-tracrRNA chimera (chimeric RNA) and SpCas9. The chimeric guide RNA contains a 20 bp guide sequence corresponding to a protospacer in the genomic target site. Figure 3B provides schematic diagrams of guide sequences targeting human EMX1, PVALB, and mouse Th loci, as well as their predicted secondary structures. The modification efficiency at each target site is shown below the RNA secondary structure diagram (EMX1, n=216 amplicon sequencing reads; PVALB, n=224 reads; Th, n=265 reads). The folding algorithm produced an output colored according to the probability that each base assumes a predicted secondary structure, as shown by the rainbow scale reproduced in grayscale in Figure 3B. Further vector designs for SpCas9 are shown in Figure 44, which describe a single-expression vector incorporating a U6 promoter bound to the insertion site for the guide oligo and a Cbh promoter bound to the SpCas9 coding sequence. The vector shown in Figure 44b contains a tracrRNA coding sequence bound to the H1 promoter.

[0151] To investigate whether spacers containing secondary structures can function in naturally functioning prokaryotic cells, transformation interference with protospacer-supported plasmids was tested in Escherichia coli (E. coli) strains heterologously expressing the Streptococcus pyogenes (S. pyogenes) SF370 CRISPR locus 1 (Figure 10). The CRISPR locus was cloned into a low-copy E. coli (E. coli) expression vector, and the crRNA array was replaced with a single spacer flanked by a DR pair (pCRISPR). E. coli (E. coli) strains possessing different pCRISPR plasmids were transformed with challenge plasmids containing the corresponding protospacer and PAM sequence (Figure 10C). In bacterial assays, all spacers promoted efficient CRISPR interference (Figure 4C). These results suggest the existence of additional factors that may influence the efficiency of CRISPR activity in mammalian cells.

[0152] To investigate the specificity of CRISPR-mediated cleavage, the effect of single nucleotide mutations in guide sequences on protospacer cleavage in mammalian genomes was analyzed using a series of EMX1-targeting chimeric crRNAs with single point mutations (Figure 4A). Figure 4B illustrates the results of a Surveyor nuclease assay comparing the cleavage efficiency of Cas9 when paired with different mutant chimeric RNAs. A single nucleotide mismatch of up to 12 bp on the 5' end of the PAM substantially halted genome cleavage by SpCas9, while spacers with mutations further upstream retained activity against the original protospacer target (Figure 4B). In addition to the PAM, SpCas9 exhibits single nucleotide specificity within the last 12 bp of the spacer. Furthermore, CRISPR can mediate genome cleavage as efficiently as a pair of TALE nucleases (TALENs) targeting the same EMX1 protospacer. Figure 4C provides a schematic diagram showing the design of a TALEN targeting EMX1, and Figure 4D shows a Surveyor gel comparing the efficiencies of TALEN and Cas9 (n=3).

[0153] We established a set of components to achieve CRISPR-mediated gene editing in mammalian cells via the error-prone NHEJ mechanism, and tested CRISPR's ability to stimulate homologous recombination (HR), a high-fidelity gene repair pathway for precise editing in the genome. Wild-type SpCas9 can mediate site-specific double-segment breaks that can be repaired through both NHEJ and HR. Furthermore, we engineered the aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of SpCas9 to convert the nuclease into a nickase (SpCas9n; explained in Figure 5A) (see, e.g., Sapranauskas et al., 2011, Nucleic Acids Research, 39:9275; Gasiunas et al., 2012, Proc. Natl. Acad. Sci. USA, 109:E2579), resulting in nicked genomic DNA undergoing high-fidelity homologous recombination repair (HDR). Surveyor assays confirmed that SpCas9n does not generate indels at the EMX1 protospacer target. As illustrated in Figure 5B, co-expression of EMX1-targeting chimeric crRNA with SpCas9 induced indels at the target site, while co-expression with SpCas9n did not (n=3). Furthermore, sequencing of 327 amplicons did not detect any indels induced by SpCas9n. CRISPR-mediated HR was tested by selecting the same locus and co-transferring HEK293FT cells with an HR template to introduce EMX1-targeting chimeric RNA, hSpCas9 or hSpCas9n, and a pair of restriction sites (HindIII and NheI) near the protospacer. Figure 5C provides a schematic explanation of the HR strategy, along with the relative localization of the recombination site and primer annealing sequences (arrows). SpCas9 and SpCas9n actually catalyzed the integration of the HR template into the EMX1 gene.PCR amplification of the target region followed by restriction digestion with HindIII revealed cleavage products corresponding to predicted fragment sizes (arrows in the restriction fragment length polymorphism gel analysis shown in Figure 5D), with SpCas9 and SpCas9n mediating similar levels of HR efficiency. The applicant further confirmed HR using Sanger sequencing of genomic amplicons (Figure 5E). These results demonstrate the utility of CRISPR for facilitating targeted gene insertions in mammalian genomes. Given the 14 bp target specificity of wild-type SpCas9 (12 bp from the spacer and 2 bp from the PAM), the availability of nickase significantly reduces the possibility of off-target modifications because the single-strand degradation products are not substrates for the error-prone NHEJ pathway.

[0154] We tested the potential of multiplexed sequence targeting by constructing an expression construct (Figure 2A) that mimics the native architecture of CRISPR loci with array spacers. Using a single CRISPR array encoding a pair of EMX1 and PVALB targeting spacers, efficient cleavage was detected at both loci (Figure 4F, showing both a schematic design of the crRNA array and a Surveyor blot demonstrating efficient cleavage mediation). Targeted deletions of larger genomic regions via simultaneous DSBs using spacers for two targets within EMX1, spaced 119 bp apart, were also tested, and a deletion efficacy of 1.6% (3 out of 182 amplicons; Figure 4G) was detected. This demonstrates that the CRISPR system can mediate multiplexed editing within a single genome.

[0155] Example 2: Modified and alternative CRISPR systems The skill of using RNA to program sequence-specific DNA cleavage defines a new class of genome engineering tools for various research and industrial applications. Several embodiments of CRISPR systems can be further improved to increase the efficiency and versatility of CRISPR targeting. Optimal Cas9 activity may depend on the availability of free Mg2+ at higher levels than that present in mammalian nuclei (see, e.g., Jinek et al., 2012, Science, 337:816), and preference for NGG motifs immediately downstream of the protospacer limits targeting ability by an average of 12 bp per target in the human genome (Figure 11, evaluating both positive and negative strands of human chromosome sequences). Some of these constraints can be overcome by leveraging the diversity of CRISPR loci across microbial metagenomes (see, e.g., Makarova et al., 2011, Nat Rev Microbiol, 9:467). Other CRISPR loci can be transplanted into the mammalian cell environment in a manner similar to that described in Example 1. For example, Figure 12 illustrates the adaptation of a type II CRISPR system from CRISPR1 to Streptococcus thermophilus LMD-9 for heterologous expression in mammalian cells to achieve CRISPR-mediated genome editing. Figure 12A provides a schematic description of CRISPR1 to S. thermophilus LMD-9. Figure 12B illustrates the design of the expression system for the S. thermophilus CRISPR system. Human codon-optimized hStCas9 is expressed using a constitutive EF1α promoter. Mature versions of tracrRNA and crRNA are expressed using a U6 promoter to facilitate accurate transcription initiation. Sequences from mature crRNA and tracrRNA are described. A single base indicated by the lowercase letter "a" in the crRNA sequence is used to remove the poly-U sequence that functions as an RNApolIII transcription terminator. Figure 12C provides schematic diagrams showing guide sequences targeting the human EMX1 locus and their predicted secondary structures.The modification efficiency at each target site is shown below the RNA secondary structure. The algorithm that generates this structure colors each base according to its probability of assuming the predicted secondary structure, which is shown in Figure 12C as a rainbow scale reproduced in grayscale. Figure 12D shows the results of hStCas9-mediated cleavage at the target locus using the Surveyor assay. RNA guide spacers 1 and 2 induced 14% and 6.4%, respectively. A statistical analysis of cleavage activity across biological replicas at these two protospacer sites is also provided in Figure 6. Figure 16 provides a schematic diagram of additional protospacers and corresponding PAM sequence targets of the S. thermophilus CRISPR system in the human EMX1 locus. The two protospacer sequences are highlighted, and their corresponding PAM sequences that satisfy the NNAGAAW motif are indicated by underlining them at the 3' end relative to the corresponding highlighted sequences. Both protospacers target the antisense strand.

[0156] Example 3: Sample Target Sequence Selection Algorithm Design a software program to identify candidate CRISPR target sequences on both strands of an input DNA sequence based on a desired guide sequence length and CRISPR motif sequence (PAM) for a given CRISPR enzyme. For example, the target site for Cas9 from Streptococcus pyogenes can be identified by searching for 5'-Nx-NGG-3' on both the input sequence and its reverse complementary strand using the PAM sequence NGG. Similarly, the target site for Cas9 from S. thermophilus CRISPR1 can be identified by searching for 5'-Nx-NNAGAAW-3' on both the input sequence and its reverse complementary strand using the PAM sequence NNAGAAW. Similarly, the target site for Cas9 in S. thermophilus CRISPR3 can be identified by searching for 5'-Nx-NGGNG-3' on both the input sequence and the reverse complementary strand of the input using the PAM sequence NGGNG. The value "x" in Nx can be fixed by the program or specified by the user, for example, 20.

[0157] Because multiple occurrences of DNA target sites in the genome can lead to nonspecific genome editing, after identifying all potential sites, the program filters out sequences based on the number of times they appear in the relevant reference genome. For CRISPR enzymes where sequence specificity is determined by the 11-12 bp 5' end of the PAM sequence, including the PAM sequence itself, the filtering step may be based on the seed sequence. Therefore, to avoid editing at additional genomic loci, the results are filtered based on the number of occurrences of the seed:PAM sequence in the relevant genome. The user can be allowed to select the length of the seed sequence. The user can also be allowed to specify the number of occurrences of the seed:PAM sequence in the genome for the purpose of passing through the filter. The default is to screen for unique sequences. The filtration level is changed by changing both the length of the seed sequence and the number of occurrences of the sequence in the genome. The program may further or alternatively provide a guide sequence complementary to the reported target sequence by providing the inverse complementary strand of the identified target sequence.

[0158] Further details of methods and algorithms for optimizing sequence selection can be found in U.S. Patent Application No. 61 / 836,080 (Agent Reference No. 44790.11.2022), which is incorporated herein by reference.

[0159] Example 4: Evaluation of multiple chimeric crRNA-tracrRNA hybrids This example describes the results obtained for chimeric RNA (chiRNA; containing a guide sequence, a tracr mate sequence, and a tracr sequence in a single transcript) having a tracr sequence that incorporates wild-type tracrRNA sequences of different lengths. Figure 18a illustrates a schematic diagram of the bicistronic expression vector for the chimeric RNA and Cas9. Cas9 is driven by the CBh promoter, and the chimeric RNA is driven by the U6 promoter. The chimeric guide RNA consists of a 20 bp guide sequence (N) bound to the truncated tracr sequence (extending from the first "U" of the lower strand to the end of the transcript) at various positions shown. The guide and tracr sequences are separated by the tracr mate sequence GUUUUAGAGCUA followed by the loop sequence GAAA. The results of SURVEYOR assays for Cas9-mediated indels at the human loci EMX1 and PVALB are shown in Figures 18b and 18c, respectively. Arrows indicate predicted SURVEYOR fragments. chiRNAs are indicated by their "+n" notation, and crRNAs refer to hybrid RNAs in which the guide and tracr sequences are expressed as separate transcripts. Quantification of these results performed in triplicates is shown as histograms in Figures 19a and 19b, corresponding to Figures 18b and 18c, respectively ("ND" indicates that no indel was detected). Table D provides the protospacer IDs and their corresponding genomic targets, protospacer sequences, PAM sequences, and chain localizations. The guide sequences were designed to be complementary to the entire protospacer sequence in the case of separate transcripts in the hybrid system, or to be complementary only to the underlined portion in the case of chimeric RNA.

[0160] [Table 18]

[0161] Cell culture and translocation Human embryonic kidney (HEK) cell line 293FT (Life Technologies) was maintained at 37°C under 5% CO2 incubation in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100 U / mL penicillin, and 100 μg / mL streptomycin. 293FT cells were seeded onto 24-well plates (Corning) at a density of 150,000 cells per well 24 hours prior to translocation. Cells were translocated using Lipofectamine 2000 (Life Technologies) according to the manufacturer's recommended protocol. A total of 500 ng of plasmid was used for each well of the 24-well plate.

[0162] SURVEYOR assay for genome modification 293FT cells were transfused with the plasmid DNA described above. The cells were incubated at 37°C for 72 hours after transfusion, and then genomic DNA was extracted. Genomic DNA was extracted using QuickExtract DNA Extraction Solution (Epicentre) according to the manufacturer's protocol. Briefly, pelletized cells were resuspended in QuickExtract solution and incubated at 65°C for 15 minutes and at 98°C for 10 minutes. Genomic regions flanking the CRISPR target sites for each gene were amplified by PCR (using the primers listed in Table E), and the products were purified using QiaQuick Spin Column (Qiagen) according to the manufacturer's protocol. A total of 400 ng of purified PCR product was mixed with 2 μl of 10× Taq DNA Polymerase PCR buffer (Enzymatics), and diluted with ultrapure water to a final volume of 20 μl. The mixture was then subjected to a re-nealing process to enable heteroduplex formation: 95°C for 10 minutes, then 95°C to 85°C with a gradient of -2°C / sec, 85°C to 25°C with a gradient of -0.25°C / sec, and finally maintained at 25°C for 1 minute. After re-nealing, the product was treated with SURVEYOR nuclease and SURVEYOR enhancer S (Transgenomics) according to the manufacturer's recommended protocol. Analysis was performed on 4–20% Novex TBE polyacrylamide gels (Life Technologies). The gels were stained with SYBR Gold DNA stain (Life Technologies) for 30 minutes and imaged using the Gel Doc gel imaging system (Bio-rad). Quantification was based on relative band intensity.

[0163] [Table 19]

[0164] Computer-aided identification of unique CRISPR target sites To identify unique target sites for the Streptococcus pyogenes (S. pyogenes) SF370Cas9 (SpCas9) enzyme in human, mouse, rat, zebrafish, fruit fly, and nematode (C. elegans) genomes, the applicants developed a software package to scan both strands of DNA sequences and identify all possible SpCas9 target sites. In this example, each SpCas9 target site was operationally defined as a 20 bp sequence followed by an NGG protospacer adjacent motif (PAM) sequence, and the applicants identified all sequences on all chromosomes that satisfy this 5'-N20-NGG-3' definition. To prevent nonspecific genome editing, after identifying all potential sites, all target sites were filtered based on the number of times they appear in the associated reference genome. For example, to utilize the sequence specificity of Cas9 activity conferred by a "seed" sequence, which can be approximately 11-12 bp from the 5' end of the PAM sequence, the 5'-NNNNNNNNNN-NGG-3' sequence was selected as unique within the relevant genome. All genome sequences were downloaded from the UCSC Genome Browser (human genome hg19, mouse genome mm9, rat genome rn5, zebrafish genome danRer7, Drosophila melanogaster genome dm4, and C. elegans genome ce10). The complete search results are available for viewing using UCSC Genome Browser information. Exemplary visualizations of some target sites in the human genome are provided in Figure 21.

[0165] First, we targeted three sites within the EMX1 locus in human HEK293FT cells. The genomic modification efficiency of each chiRNA was evaluated using the SURVEYOR nuclease assay, which detects mutations resulting from subsequent repair by the DNA double-strand break (DSB) and non-homologous end-joining (NHEJ) DNA damage repair pathways. Constructs denoted as chiRNA(+n) indicate that the chimeric RNA construct contains up to +n nucleotides of wild-type tracrRNA, with values ​​of 48, 54, 67, and 85 used for n. Chimeric RNAs containing longer fragments of wild-type tracrRNA (chiRNA(+67) and chiRNA(+85)) mediated DNA cleavage at all three EMX1 target sites, with chiRNA(+85) in particular demonstrating significantly higher levels of DNA cleavage than the corresponding crRNA / tracrRNA hybrids expressing the guide and tracr sequences in separate transcripts (Figures 18b and 19a). Two sites within the PVALB locus that did not exhibit detectable cleavage in the hybrid system (guide and tracr sequences expressed as separate transcripts) were also targeted using chiRNA. chiRNA(+67) and chiRNA(+85) were able to mediate significant cleavage in the two PVALB protospacers (Figures 18c and 19b).

[0166] A consistent increase in genome modification efficiency was observed with increasing tracr sequence length for all five targets in the EMX1 and PVALB loci. While not constrained by any theory, the secondary structure formed by the 3' end of the tracrRNA may play a role in improving the rate of CRISPR complex formation. A description of the predicted secondary structure for each of the chimeric RNAs used in this embodiment is provided in Figure 21. The secondary structures were predicted using RNAfold (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAfold.cgi), which uses the minimum free energy and partition function algorithm. Pseudocolor (reproduced in grayscale) for each base indicates the probability of pair formation. Since chiRNAs with longer tracr sequences were able to cleave targets that were not cleaved by the natural CRISPRcrRNA / tracrRNA hybrid, it is conceivable that chimeric RNAs can be loaded onto Cas9 more efficiently than their natural hybrid equivalents. To facilitate the application of Cas9 for site-directed genome editing in eukaryotic cells and organisms, all predicted unique target sites for Streptococcus pyogenes (S. pyogenes) Cas9 were computer-aided in the genomes of humans, mice, rats, zebrafish, nematodes (C. elegans), and Drosophila melanogaster (D. melanogaster). Chimeric RNAs can be designed for Cas9 enzymes from other microorganisms to expand the target space for CRISPR RNA programmable nucleases.

[0167] Figure 22 shows the wild-type tracrRNA sequence up to +85 nucleotides, and nuclear localization. Exemplary bicistronic expression for chimeric RNA containing SpCas9 with columns Let's explain the expression vector. SpCas9 is expressed from the CBh promoter and bGH It is terminated by the rh-A signal (bGHpA). The enlarged sequence explained directly below the schematic diagram is: Corresponding to the region surrounding the guide sequence insertion site, from 5' to 3' it includes the 3' portion of the U6 promoter (the first shaded region), the BbsI cleavage site (arrow), the partial direct repeat (tracr mate sequence GTTTTAGAGCTA, underlined), the loop sequence GAAA, and the +85 tracr sequence (the underlined sequence after the loop sequence). An exemplary guide sequence insert is illustrated below the guide sequence insertion site, and the nucleotides of the guide sequence for the selected target are represented by "N".

[0168] The sequences described in the above examples are as follows (the polynucleotide sequences are from 5' to 3'): U6 - short tracrRNA (Streptococcus pyogenes SF370):

Chem.

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Chem.

Chem.

[0169] Example 5: RNA-guided editing of bacterial genomes using the CRISPR-Cas system The applicants introduced precise mutations into the genomes of Streptococcus pneumoniae and Escherichia coli using the CRISPR-associated endonuclease Cas9. This approach relies on Cas9-directed cleavage at targeted sites to kill non-mutant cells, avoiding the need for selectable markers or counter-selection systems. Cas9 specificity was reprogrammed by altering short CRISPR RNA (crRNA) sequences to produce single and multinucleotide changes supported on the editing template. The simultaneous use of two crRNAs enabled the introduction of multiple mutations. In Streptococcus pneumoniae, nearly 100% of cells surviving Cas9 cleavage contained the desired mutation, and in E. coli, when used in combination with repairing, 65% contained the desired mutation. The applicants primarily focused on thoroughly analyzing Cas9 targeting requirements to define the range of targetable sequences and providing guidelines for editing sites that do not meet those requirements, which suggests the versatility of this technology for bacterial genome engineering.

[0170] Understanding gene function depends on the possibility of intracellular DNA sequence changes in regulatory modes. Site-directed mutagenesis in eukaryotes is achieved by using sequence-specific nucleases that promote homologous recombination of template DNA containing the target mutation. Zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and homing meganucleases can be programmed to cleave genomes at specific localizations, but these approaches require the engineering of novel enzymes for their respective target sequences. In prokaryotes, mutagenesis methods require a two-step process involving the introduction of a selection marker into the locus to be edited or a counter-selection system. In recent years, phage recombinant proteins, a technique that promotes homologous recombination of linear DNA or oligonucleotides, have been used for repair. However, repair efficiency can be relatively low due to the absence of mutation selection (0.1-10% for point mutations leading to 10⁻⁵-10⁻⁶ for larger modifications), and often requires screening of a large number of colonies. Therefore, there is still a need for new technologies that are inexpensive, easy to use, and efficient for gene engineering in both eukaryotes and prokaryotes.

[0171] Recent studies on the CRISPR (Clustered Equispaced Short Repeat) adaptive immune system in prokaryotes have led to the identification of nucleases whose sequence specificity is programmed by small RNA molecules. The CRISPR locus consists of a series of repeats separated by "spacer" sequences that match the genomes of bacteriophages and other mobile gene elements. The repeat-spacer array is transcribed as a long precursor, generating small crRNA molecules that define target sequences (also known as protospacers) that are processed within the repeat sequences and cleaved by the CRISPR system. The presence of a sequence motif immediately downstream of a known target region, such as a protospacer adjacency motif (PAM), is essential for cleavage. CRISPR-related (cas) genes typically encode enzymatic mechanisms responsible for flanking the repeat-spacer array and for crRNA biogenesis and targeting. Cas9 is a dsDNA endonuclease that uses crRNA guidance to define the cleavage site. The loading of crRNA guides onto Cas9 occurs during the processing of crRNA precursors and requires small RNA antisense, tracrRNA, and RNase III against the precursor. In contrast to genome editing with ZFNs or TALENs, altering Cas9 target specificity requires only the design of short crRNA guides, rather than protein engineering.

[0172] The applicants have recently shown that the introduction of a CRISPR system targeting a chromosomal locus in Streptococcus pneumoniae results in the killing of transformed cells. Accidental survivors were observed to contain mutations in the target region, suggesting that Cas9 dsDNA endonuclease activity against endogenous targets can be used for genome editing. The applicants have shown that markerless mutations can be introduced through transformation of a template DNA fragment that recombinates in the genome and eliminates Cas9 target recognition. The directing of Cas9 specificity by several different crRNAs allows for the simultaneous introduction of multiple mutations. The applicants have also characterized the sequence requirements for Cas9 targeting in detail and demonstrate that this approach can be combined with reconciliation for genome editing in Escherichia coli (E. coli).

[0173] Results: Genome editing via Cas9 cleavage of chromosome targets The Streptococcus pneumoniae (S. pneumoniae) strain crR6 contains a Cas9-based CRISPR system that cleaves a target sequence present in the bacteriophage φ8232.5. This target was integrated into the srtA chromosomal locus of a second strain, R68232.5. A variant target sequence containing a mutation in the PAM region was integrated into the srtA locus of a third strain, R6370.1, making this strain "immune" to CRISPR cleavage (Figure 28a). The applicants transformed R68232.5 and R6370.1 cells with genomic DNA from crR6 cells and predicted that successful transformation of R68232.5 cells should result in target locus cleavage and cell death. Contrary to this prediction, the applicants isolated R68232.5 transformants with approximately 10 times lower efficiency than R6370.1 transformants (Figure 28b). Genetic analysis of eight R68232.5 transformants (Figure 28) revealed that the majority were products of a double recombination event that eliminated the toxicity of Cas9 targeting by replacing the φ8232.5 target with the crR6 genome wild-type srtA locus, which does not contain the protospacer required for Cas9 recognition. These results demonstrated that the simultaneous introduction of a CRISPR system that targets a genomic locus (targeting construct) together with a template for recombination into the targeted locus (editing template) results in targeted genome editing (Figure 23a).

[0174] To create a simplified genome editing system, the applicants modified the CRISPR locus in strain crR6 by deleting the cas1, cas2, and csn2 genes, which have been shown to be non-essential for CRISPR targeting, to produce strain crR6M (Figure 28a). This strain retained the same characteristics as crR6 (Figure 28b). To demonstrate that the efficiency of Cas9-based editing can be increased and the introduced mutations can be controlled using an optimal DNA template, the applicants co-transformed R68232.5 cells with PCR products targeting either the wild-type srtA gene or the mutant R6370.1, either of which should be resistant to Cas9 cleavage. This resulted in a 5 to 10-fold increase in transformation frequency compared to genomic crR6DNA alone (Figure 23b). The editing efficiency also increased substantially, with all eight transformants tested containing wild-type srtA copies, and seven of the eight containing PAM mutations present in the R6370.1 target (Figures 23b and 29a). In summary, these results demonstrate the potential of Cas9-assisted genome editing.

[0175] Analysis of Cas9 Targeting Requirements: To introduce a specific change in the genome, an editing template must be used that carries a mutation that halts Cas9-mediated cleavage and thereby prevents cell death. This is easily achieved when a deletion of the target or its replacement with another sequence (gene insertion) is required. When the objective is to produce a gene fusion or generate a single nucleotide mutation, halting Cas9 nuclease activity is only possible by introducing a mutation in the editing template that alters either the PAM or protospacer sequence. To determine the constraints of CRISPR-mediated editing, the applicants conducted a thorough analysis of PAM and protospacer mutations that halt CRISPR targeting.

[0176] Previous studies have suggested that *Streptococcus pyogenes* Cas9 requires NGG PAM immediately downstream of the protospacer. However, since only a very limited number of PAM-inactivating mutations have been described to date, we conducted a systematic analysis to identify all post-protospacer 5-nucleotide sequences that exclude CRISPR cleavage. We generated all 1,024 possible PAM sequences in heterologous PCR products transformed into crR6 or R6 cells using randomized oligonucleotides. Constructs carrying functional PAMs were predicted to be recognized and disrupted by Cas9 in crR6 cells rather than R6 cells (Figure 24a). Colonies exceeding 2 × 10⁵ were pooled together to extract DNA to be used as templates for simultaneous amplification of all targets. The PCR products were deep-sequenced and found to contain all 1,024 sequences, with coverage ranging from 5 to 42,472 reads (see Section “Analysis of Deep Sequencing Data”). The functionality of each PAM was estimated by the relative ratio of its reads in the crR6 sample to that of the R6 sample. Analysis of the first three bases of the PAM, averaging the last two bases, clearly showed that the NGG pattern was underrepresented in the crR6 transformant (Figure 24b). Furthermore, the next two bases had no detectable effect on the NGG PAM (see section “Analysis of Deep Sequencing Data”), demonstrating that the NGGNN sequence was sufficient to tolerate Cas9 activity. Partial targeting was observed for the NAG PAM sequence (Figure 24b). In addition, the NNGGN pattern partially inactivated CRISPR targeting (Table G), indicating that the NGG motif could still be recognized by Cas9 with reduced efficiency even when shifted by only 1 bp. These data highlight the molecular mechanism of Cas9 target recognition, revealing that the NGG (or CCN on the complementary chain) sequence is sufficient for Cas9 targeting and that mutations from NGG to NAG or NNGGN in the editing template should be avoided.Due to the high frequency of these trinucleotide sequences (once every 8 bp), this means that editing can be performed at almost any location in the genome. In fact, the applicants tested 10 randomly selected targets carrying various PAMs and found all to be functional (Figure 30).

[0177] Another method to disrupt Cas9-mediated cleavage is to introduce mutations in the protospacer region of the editing template. It is known that point mutations within the "seed sequence" (8 to 10 protospacer nucleotides directly adjacent to the PAM) can halt CRISPR nuclease-mediated cleavage. However, the exact length of this region is unknown, and it is unclear which nucleotide mutations in the seed can disrupt Cas9 target recognition. We randomized the entire protospacer sequence involved in base pairing with crRNA according to the same deep sequencing approach described above to determine all sequences that disrupt targeting. We randomized the positions of each of the 20 matching nucleotides (14) in the spc1 target present in R68232.5 cells (Figure 23a) and transformed them into crR6 and R6 cells (Figure 24a). Consistent with the presence of the seed sequence, only mutations in 12 nucleotides immediately upstream of the PAM halted Cas9-mediated cleavage (Figure 24c). However, different mutations showed significantly different effects. The distal positions of the seed (from PAM) (positions 12 to 7) tolerated most mutations, with only one specific base substitution halting targeting. In contrast, any nucleotide mutation at the proximal positions (from 6 to 1, excluding position 3) eliminated Cas9 activity, albeit at different levels for each specific substitution. At position 3, only two substitutions affected CRISPR activity, with varying intensities. We conclude that while seed sequence mutations can interfere with CRISPR targeting, there are limitations on the nucleotide changes that can be made at each position of the seed. Furthermore, these limitations are most likely to be varied by different spacer sequences. Therefore, we believe that mutations in the PAM sequence should be the preferred editing policy, if possible. Alternatively, multiple mutations in the seed sequence can be introduced to interfere with Cas9 nuclease activity.

[0178] Cas9-mediated genome editing in Streptococcus pneumoniae (S. pneumonia): To develop a rapid and efficient method for targeted genome editing, we engineered strain crR6Rk, a strain in which a spacer can be easily introduced by PCR (Figure 33). We decided to edit the β-galactosidase (bgaA) gene of Streptococcus pneumoniae (S. pneumonia) in which activity can be easily measured. We introduced alanine substitutions of amino acids in the active site of this enzyme: R481A (R→A) and N563A, E564A (NE→AA) mutations. To illustrate different editing strategies, we designed mutations in both the PAM sequence and the protospacer seed. In both cases, we used the same targeting construct having a complementary crRNA in the region of the β-galactosidase gene adjacent to the TGG PAM sequence (CCA in the complementary strand, Figure 26). The R→A editing template created a 3-nucleotide mismatch on the protospacer seed sequence (CGT to GCA, also introducing the BtgZI restriction site). In the NE→AA editing template, the applicants simultaneously introduced a synonymous mutation (TGG to TTG) that creates an inactive PAM, along with a 218nt downstream mutation of the protospacer region (AAT GAA to GCT GCA, also generating the TseI restriction site). This final editing strategy demonstrated the potential to create mutations in locations where selecting an appropriate target using distant PAMs might be difficult. For example, the Streptococcus pneumoniae (S. pneumoniae) R6 genome has a 39.7% GC content and contains an average of one PAM motif every 12 bp, although some PAM motifs are separated by up to 194 bp (Figure 33). Furthermore, the applicants designed a 6,664 bp ΔbgaA in-frame deletion. In all three cases, simultaneous transformation with the targeting and editing templates produced cells that were 10 times more kanamycin-resistant than simultaneous transformation with a control editing template containing the wild-type bgaA sequence (Figure 25b). The applicants genotyped 24 transformants (8 for each editing experiment) and found that all but one incorporated the desired changes (Figure 25c).DNA sequencing confirmed not only the presence of introduced mutations but also the absence of secondary mutations in the target region (Figure 29b, c). Finally, the applicants confirmed that all edited cells exhibited the predicted phenotype by measuring β-galactosidase activity (Figure 25d).

[0179] Multiple mutations can also be generated for the study of biological pathways using Cas9-mediated editing. The applicant decided to describe a saltase-dependent pathway that immobilizes surface proteins on the envelope of Gram-positive bacteria. The applicant introduced saltase deletion by simultaneous transformation of a chloramphenicol-resistant targeting construct and a ΔsrtA editing template (Figure 33a, b), followed by ΔbgaA deletion using a kanamycin-resistant targeting construct replacing ΔbgaA. In Streptococcus pneumoniae, β-galactosidase is covalently bound to the cell wall by saltase. Therefore, srtA deletion results in the release of surface proteins into the supernatant, while double deletion results in the absence of detectable β-galactosidase activity (Figure 34c). Such sequential selection can be repeated as many times as needed to generate multiple mutations.

[0180] These two mutations can also be introduced simultaneously. The applicants designed a targeting construct containing two spacers, one matching srtA and the other matching bgaA, and co-transformed it with both editing templates simultaneously (Figure 25e). Genetic analysis of the transformants showed that editing occurred in 6 out of 8 cases (Figure 25f). In particular, the remaining two clones contained either ΔsrtA or ΔbgaA deletion, respectively, which suggested the possibility of performing combinatorial mutagenesis using Cas9. Finally, to eliminate CRISPR sequences, the applicants introduced a plasmid containing the bgaA target and spectinomycin resistance gene along with genomic DNA from wild-type strain R6. Spectinomycin-resistant transformants carrying the plasmid eliminated CRISPR sequences (Figures 34a, d).

[0181] Editing Mechanism and Efficiency: To understand the underlying mechanism of Cas9-mediated genome editing, we designed an experiment to measure editing efficiency independently of Cas9 cleavage. We integrated the ermAM erythromycin resistance gene into the srtA locus and introduced an early stop codon using Cas9-mediated editing (Figure 33). The resulting strain (JEN53) contained the ermAM(stop) allele and was sensitive to erythromycin. Using this strain, the efficiency of ermAM gene repair could be evaluated by measuring the rate at which antibiotic resistance was restored in cells with or without Cas9 cleavage. JEN53 was transformed with either a kanamycin-resistant CRISPR construct targeting the ermAM(stop) allele (CRISPR::ermAM(stop)) or a control construct without a spacer (CRISPR::φ) using an editing template that restored the wild-type allele (Figure 26a, b). In the absence of kanamycin selection, the rate of edited colonies was on the order of 10⁻² (erythromycin-resistant cfu / total cfu) (Figure 26c), which represented the baseline frequency of recombination without Cas9-mediated selection over unedited cells. However, when kanamycin selection was applied and the control CRISPR construct was co-transformed, the rate of edited colonies increased to approximately 10⁻¹ (kanamycin and erythromycin-resistant cfu / kanamycin-resistant cfu) (Figure 26c). This result indicates that selection of recombination at the CRISPR locus co-selected recombination at the ermAM locus independently of Cas9 cleavage of the genome, suggesting that a subpopulation of cells is prone to transformation and / or recombination. Transformation with the CRISPR::ermAM(terminate) construct followed by kanamycin selection resulted in an increase to 99% of the rate of erythromycin-resistant edited cells (Figure 26c). To determine whether this increase is caused by the killing of non-edited cells, the applicants compared kanamycin-resistant colony-forming units (cfus) obtained after co-transformation of JEN53 cells with CRISPR::ermAM(terminate) or CRISPR::φ constructs.

[0182] The applicants counted 5.3 times fewer kanamycin-resistant colonies after transformation with the ermAM(stop) construct (2.5×10⁴ / 4.7×10³, Figure 35a), suggesting that Cas9-mediated targeting of chromosomal loci actually leads to the killing of non-edited cells. Finally, since it is known that the introduction of dsDNA degradation in bacterial chromosomes triggers repair mechanisms that increase the rate of recombination of damaged DNA, the applicants investigated whether Cas9-mediated cleavage induces recombination of the editing template. The applicants counted 2.2 times more colonies after co-transformation with the CRISPR::erm(stop) construct than with the CRISPR::φ construct (Figure 26d), indicating the presence of moderate recombination induction. In summary, these results demonstrate that simultaneous selection of transformable cells, induction of recombination by Cas9-mediated cleavage, and selection of non-edited cells each contribute to highly efficient genome editing in Streptococcus pneumoniae.

[0183] Because Cas9-mediated genome cleavage kills non-edited cells, it is predicted that no cells that accepted the kanamycin-resistant Cas9 cassette will be recovered, except for those that did not accept the editing template. However, in the absence of the editing template, the applicant recovered many kanamycin-resistant colonies after transformation with the CRISPR::ermAM(terminate) construct (Figure 35a). These cells, which "escape" CRISPR-induced death, produced background that determined the limitations of the method. This background frequency can be calculated as the ratio of CRISPR::ermAM(terminate) / CRISPR::φcfu, which in this experiment was 2.6 × 10⁻³ (7.1 × 10¹ / 2.7 × 10⁴), meaning that if the recombination frequency of the editing template is less than this value, CRISPR selection may not be able to efficiently recover the desired mutants above the background. To understand the origin of these cells, we genotyped eight background colonies and found that seven lacked a targeting spacer (Figure 35b) and one harbored a putative inactivating mutation in Cas9 (Figure 35c).

[0184] Genome editing with Cas9 in Escherichia coli (E. coli): Activation of Cas9 targeting through chromosomal integration of the CRISPR-Cas system is only possible in highly recombinant organisms. To develop a more general method applicable to other microorganisms, we decided to perform genome editing in Escherichia coli (E. coli) using a plasmid-based CRISPR-Cas system. Two plasmids were constructed: a pCas9 plasmid carrying tracrRNA, Cas9, and a chloramphenicol resistance cassette (Figure 36), and a pCRISPR kanamycin resistance plasmid carrying an array of CRISPR spacers. To measure the efficiency of editing independent of CRISPR selection, we sought to introduce an A-to-C transversion in the rpsL gene that confers streptomycin resistance. The applicants constructed a pCRISPR::rpsL plasmid containing a spacer that guides Cas9 cleavage of the wild-type rpsL allele but not of the mutant rpsL allele (Figure 27b). The pCas9 plasmid was first introduced into Escherichia coli (E. coli) MG1655, and the resulting strain was co-transformed with the pCRISPR::rpsL plasmid and edited oligonucleotide W542 containing the A-to-C mutation. Only streptomycin-resistant colonies were recovered after transformation with the pCRISPR::rpsL plasmid, suggesting that Cas9 cleavage induces oligonucleotide recombination (Figure 37). However, the number of streptomycin-resistant colonies was two orders of magnitude lower than the number of kanamycin-resistant colonies, which are presumably cells that escape Cas9 cleavage. Therefore, under these conditions, Cas9 cleavage promoted the introduction of mutations, but the efficiency was not sufficient to select mutant cells over the background of "escapers".

[0185] To improve the efficiency of genome editing in Escherichia coli (E. coli), the applicants applied the CRISPR system of the present invention by reconciliation using Cas9-induced cell death to select desired mutations. The pCas9 plasmid was introduced into the reconciliation strain HME63(31) containing Gam, Exo, and Beta functions of the □-red phage. The resulting strain was co-transformed with the pCRISPR::rpsL plasmid (or pCRISPR::φ control) and W542 oligonucleotide (Figure 27a). The reconciliation efficiency was 5.3 × 10⁻⁵, calculated as the percentage of total cells resistant to streptomycin when using the control plasmid (Figure 27c). In contrast, transformation with the pCRISPR::rpsL plasmid increased the percentage of mutant somatic cells to 65 ± 14% (Figures 27c and 29f). The applicants observed that the number of cfus was reduced by approximately three orders of magnitude after transformation with the pCRISPR::rpsL plasmid compared to the control plasmid (4.8 × 10⁵ / 5.3 × 10ⁿ, Figure 38a), suggesting that the selection resulted from CRISPR-induced death in unedited cells. To measure the ratio of Cas9 cleavage inactivation, a key parameter of the applicants' method, the applicants transformed cells with either the pCRISPR::rpsL or control plasmid without using W542-edited oligonucleotides (Figure 38a). This background for CRISPR "escapers," measured as the pCRISPR::rpsL / pCRISPR::φcfu ratio, was 2.5 × 10⁻⁴ (1.2 × 10ⁿ / 4.8 × 10⁵). Eight genotyping of these escapers revealed the presence of targeting spacer deletion in all cases (Figure 38b). This background was higher than the repair efficiency of the rpsL mutation (5.3 × 10⁻⁵), suggesting that Cas9 cleavage must induce oligonucleotide recombination to obtain 65% edited cells. To confirm this, we compared the number of kanamycin and streptomycin-resistant cfus after transformation of pCRISPR::rpsL or pCRISPR::φ (Figure 27d).Similar to the case of Streptococcus pneumoniae, the applicants observed a moderate induction of recombination of approximately 6.7 times (2.0 × 10⁻⁴ / 3.0 × 10⁻⁵). In summary, these results demonstrate that the CRISPR system provides a method for selecting mutations introduced by repair.

[0186] The applicants have shown that the CRISPR-Cas system can be used for targeted genome editing in bacteria by simultaneous transformation of a targeting construct that kills wild-type cells and an editing template that eliminates CRISPR cleavage and induces desired mutations. Different types of mutations (insertions, deletions, or scarless single-nucleotide substitutions) can be generated. Multiple mutations can be introduced simultaneously. The specificity and versatility of editing using the CRISPR system depend on several unique properties of the Cas9 endonuclease: (i) its target specificity can be programmed by small RNA molecules without the need for enzyme engineering; (ii) its target specificity is extremely high, determined by a 20 bp RNA-DNA interaction, with a low probability of non-target recognition; (iii) almost any sequence can be targeted, with the only requirement being the presence of an adjacent NGG sequence; and (iv) almost any mutation in the NGG sequence, and mutations in the protospacer seed sequence, exclude targeting.

[0187] The applicants have demonstrated that genome engineering using the CRISPR system functions not only in highly recombinant bacteria, such as Streptococcus pneumoniae, but also in Escherichia coli (E. coli). The results in E. coli suggest that the method may be applicable to other microorganisms into which plasmids can be introduced. In E. coli, this approach complements the reconciliation of mutagenic oligonucleotides. To use this methodology in microorganisms where reconciliation is not possible, the host homologous recombination mechanism can be used by providing an editing template on a plasmid. Furthermore, accumulated evidence shows that CRISPR-mediated chromosome cleavage leads to cell death in many bacteria and archaea, making it possible to envision the use of the endogenous CRISPR-Cas system for editing purposes.

[0188] In both Streptococcus pneumoniae and Escherichia coli, the applicants observed that editing is facilitated by the simultaneous selection of transformable cells and minimal induction of recombination at the target site via Cas9 cleavage, but the mechanism contributing most to editing is selection of non-edited cells. Therefore, a major limitation of the method was the presence of a background of cells that escape CRISPR-induced cell death and lack the desired mutations. The applicants showed that these “escapers” arise primarily through the deletion of targeting spacers after recombination of repeat sequences that presumably flank the targeting spacers. Further improvements could focus on engineering flanking sequences that are sufficiently different from each other to eliminate recombination, although the biogenesis of functional crRNAs may still be supported. Alternatively, direct transformation of chimeric crRNAs could be investigated. In the specific case of Escherichia coli, the construction of the CRISPR-Cas system was not possible when this organism was also used as a cloning host. The applicants solved this problem by placing Cas9 and tracrRNA on a plasmid different from the CRISPR array. Engineering of the induction system can also circumvent this limitation.

[0189] While new DNA synthesis technologies offer the ability to create arbitrary sequences cost-effectively and with high throughput, the difficulty remains in integrating synthetic DNA into living cells to create a functional genome. Recently, the simultaneous selection MAGE strategy has been demonstrated to improve the mutation efficiency of restoration by selecting a subpopulation of cells with an increased likelihood of achieving recombination at or around a given locus. In this method, the introduction of selectable mutations increases the opportunity to generate nearby non-selectable mutations. In contrast to the indirect selection provided by this strategy, the use of CRISPR systems makes it possible to directly select desired mutations and recover them with high efficiency. These technologies expand the toolbox of genetic engineers, and together with DNA synthesis, they can substantially advance the skills to decipher gene function and manipulate organisms for biotechnological purposes. Two other studies also concern CRISPR-assisted engineering of mammalian genomes. These crRNA-directed genome editing technologies are expected to have broad applications in basic and medical sciences.

[0190] Strain and culture conditions. Streptococcus pneumoniae strain R6 was provided by Dr. Alexander Tomasz. Strain crR6 was generated in the above test. Liquid cultures of Streptococcus pneumoniae were grown in THYE medium (30 g / l Todd-Hewitt agar, 5 g / l yeast extract). Cells were plated on trypsin soybean agar (TSA) supplemented with 5% defibrinated sheep blood. Antibiotics were added as appropriate, as follows: kanamycin (400 μg / ml), chloramphenicol (5 μg / ml), erythromycin (1 μg / ml), streptomycin (100 μg / ml), or spectinomycin (100 μg / ml). β-galactosidase activity was measured using the Miller assay as described above.

[0191] Escherichia coli (E. coli) strains MG1655 and HME63 (derived from MG1655, Δ(argF-lac)U169λcI857Δcro-bioA galK tyr145UAG mutS<>amp)(31) were provided by Jeff Roberts and Donald Court, respectively. Liquid cultures of Escherichia coli (E. coli) were grown in LB medium (Difco). Antibiotics were added as appropriate, as follows: chloramphenicol (25 μg / ml), kanamycin (25 μg / ml), and streptomycin (50 μg / ml).

[0192] Streptococcus pneumoniae transformation. Competent cells were prepared as described above (23). For all genome editing transformations, the cells were gently thawed on ice and resuspended in 10 volumes of M2 medium supplemented with 100 ng / ml of competence-stimulating peptide CSP1 (40), and then the editing construct was added (the editing construct was added to the cells at a final concentration of 0.7 ng / μl to 2.5 μg / µl). The cells were incubated at 37°C for 20 minutes, then 2 μl of the targeting construct was added, and then incubated at 37°C for 40 minutes. Serial dilutions of the cells were plated on appropriate media and the colony-forming unit (cfu) count was measured.

[0193] E. coli (Lambda-red) reconciliation. Strain HME63 was used for all reconciliation experiments. Reconciliation cells were prepared and handled according to a previously published protocol (6). Briefly, 2 ml of overnight culture (LB medium) inoculated from a single colony obtained from a plate was grown at 30°C. The overnight culture was diluted 100-fold and grown at 30°C with shaking (200 rpm) until the OD600 reached 0.4-0.5 (approximately 3 hours). To induce lambda-red, the culture was transferred to a 42°C water bath and shaken at 200 rpm for 15 minutes. Immediately after induction, the culture was rotated in an ice water slurry and refrigerated on ice for 5-10 minutes. The cells were then washed and aliquoted according to the protocol. For electrotransformation, 50 μl of cells were mixed with 1 mM salt-free oligonucleotide (IDT) or 100–150 ng of plasmid DNA (prepared by QIAprep Spin Miniprep Kit, Qiagen). The cells were electroporated using a 1 mm Gene Pulser cuvette (Bio-rad) at 1.8 kV and immediately resuspended in 1 ml of room temperature LB medium. The cells were harvested at 30°C for 1–2 hours, plated on LB agar with appropriate antibiotic resistance, and incubated overnight at 32°C.

[0194] Preparation of Streptococcus pneumoniae (S. pneumoniae) genomic DNA. For transformation purposes, Streptococcus pneumoniae (S. pneumoniae) genomic DNA was extracted using the Wizard Genomic DNA Purification Kit according to the instructions provided by the manufacturer (Promega). For genotyping purposes, 700 µl of overnight Streptococcus pneumoniae (S. pneumoniae) culture was pelleted, resuspended in 60 µl of lysozyme solution (2 mg / ml), and incubated at 37°C for 30 minutes. Genomic DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen).

[0195] Strain construction. All primers used in this study are provided in Table G. An intermediate strain, LAM226, was prepared to generate *Streptococcus pneumoniae* crR6M. In this strain, the aphA-3 gene (providing kanamycin resistance) adjacent to the CRISPR array of *Streptococcus pneumoniae* crR6 strain was replaced with the cat gene (providing chloramphenicol resistance). Briefly, crR6 genomic DNA was amplified using primers L448 / L444 and L447 / L481, respectively. The cat gene was amplified from plasmid pC194 using primers L445 / L446. Each PCR product was gel-purified, and all three were fused by SOEing PCR using primers L448 / L481. The resulting PCR products were transformed into competent *Streptococcus pneumoniae* crR6 cells, and chloramphenicol-resistant transformants were selected. To generate *Streptococcus pneumoniae* crR6M, *Streptococcus pneumoniae* crR6 genomic DNA was amplified by PCR using primers L409 / L488 and L448 / L481, respectively. Each PCR product was gel-purified and fused using SOEing PCR with primers L409 / L481. The resulting PCR products were used to transform competent *Streptococcus pneumoniae* LAM226 cells, and kanamycin-resistant transformants were selected.

[0196] To generate *Streptococcus pneumoniae* crR6Rc, *Streptococcus pneumoniae* crR6M genomic DNA was amplified by PCR using primers L430 / W286, and *Streptococcus pneumoniae* LAM226 genomic DNA was amplified by PCR using primers W288 / L481. Each PCR product was gel-purified, and they were fused using SOEing PCR with primers L430 / L481. The resulting PCR products were used to transform competent *Streptococcus pneumoniae* crR6M cells, and chloramphenicol-resistant transformants were selected.

[0197] To generate *Streptococcus pneumoniae* crR6Rk, *Streptococcus pneumoniae* crR6M genomic DNA was amplified by PCR using primers L430 / W286 and W287 / L481, respectively. Each PCR product was gel-purified, and they were fused using primer L430 / L481 via SOEing PCR. The resulting PCR products were then transformed into competent *Streptococcus pneumoniae* crR6Rc cells, and kanamycin-resistant transformants were selected.

[0198] To generate JEN37, *Streptococcus pneumoniae* crR6Rk genomic DNA was amplified by PCR using primers L430 / W356 and W357 / L481, respectively. Each PCR product was gel-purified and fused using primer L430 / L481 via SOEing PCR. The resulting PCR products were then transformed into competent *Streptococcus pneumoniae* crR6Rc cells, and kanamycin-resistant transformants were selected.

[0199] To generate JEN38, R6 genomic DNA was amplified using primers L422 / L461 and L459 / L426, respectively. The ermAM gene (which defines erythromycin resistance) was amplified from plasmid pFW1543 using primers L457 / L458. Each PCR product was gel-purified, and all three were fused by SOEing PCR using primers L422 / L426. The resulting PCR products were transformed into competent Streptococcus pneumoniae (S. pneumoniae) crR6Rc cells, and erythromycin-resistant transformants were selected.

[0200] Streptococcus pneumoniae (S. pneumoniae) JEN53 was generated in two steps. First, JEN43 was constructed as described in Figure 33. JEN53 was generated by transforming competent JEN43 cells with the genomic DNA of JEN25 and selecting them based on both chloramphenicol and erythromycin.

[0201] To generate *Streptococcus pneumoniae* JEN62, *Streptococcus pneumoniae* crR6Rk genomic DNA was amplified by PCR using primers W256 / W365 and W366 / L403, respectively. Each PCR product was purified and ligated by Gibson assembly. The assembly product was transformed into competent *Streptococcus pneumoniae* crR6Rc cells, and kanamycin-resistant transformants were selected.

[0202] Plasmid construction. pDB97 was constructed via phosphorylation and annealing of oligonucleotides B296 / B297, followed by ligation into pLZ12spec digested with EcoRI / BamHI. The applicants fully sequenced pLZ12spec and deposited its sequence in GeneBank (accession number: KC112384).

[0203] pDB98 was obtained after cloning the CRISPR reader sequence together with a repeat-spacer-repeat unit into pLZ12spec. This was achieved through amplification of crR6RcDNA using primers B298 / B320 and B299 / B321, followed by SOEing PCR of both products and cloning into pLZ12spec with the restriction site BamHI / EcoRI. Thus, the spacer sequence in pDB98 was engineered to contain two BsaI restriction sites in opposite directions, enabling scarless cloning of a novel spacer.

[0204] pDB99 to pDB108 was constructed by ery ringing of oligonucleotides B300 / B301 (pDB99), B302 / B303 (pDB100), B304 / B305 (pDB101), B306 / B307 (pDB102), B308 / B309 (pDB103), B310 / B311 (pDB104), B312 / B313 (pDB105), B314 / B315 (pDB106), B315 / B317 (pDB107), and B318 / B319 (pDB108), followed by ligation in pDB98 cleaved with BsaI.

[0205] The pCas9 plasmid was constructed as follows: Essential CRISPR elements were added. , using a flanking homology arm for Gibson assembly, Streptococcus pyogenes (Streptococcos pyogenes) Amplified from SF370 genomic DNA TracrRNA and Cas9 were amplified using oligonucleotides HC008 and HC010. The leader and CRISPR sequences were amplified using HC011 / HC014 and HC015 / HC009 so that two BsaI IIS-type sites were introduced between two direct repeats to facilitate the insertion of spacers.

[0206] The pCRISPR array was constructed by subcloning the pCas9CRISPR array in pZE21-MCS1 through amplification using oligo B298+B299 and restriction using EcoRI and BamHI. The rpsL targeting spacer was cloned by alliling oligo B352+B353 and cloning it into the BsaI-cleaved pCRISPR array, thereby obtaining pCRISPR::rpsL.

[0207] Targeting and editing construct generation. Targeting constructs used for genome editing were prepared by Gibson assembly of left-hand and right-hand PCR (Table G). Editing constructs were prepared by SOEing PCR, where applicable, by fusing PCR product A (PCR A), PCR product B (PCR B), and PCR product C (PCR C) (Table G). CRISPR::φ and CRISPR::ermAM (stop) targeting constructs were prepared by PCR amplification using oligos L409 and L481 of JEN62 and crR6 genomic DNA, respectively.

[0208] Target generation using randomized PAM or protospacer sequences. The 5 nucleotides after the Spacer 1 target were randomized through amplification of R68232.5 genomic DNA using primer W377 / L426. This PCR product was then assembled with the cat gene and srtA upstream region amplified from the same template using primer L422 / W376. Strains R6 and crR6 were transformed using 80 ng of assembled DNA. Samples for randomization were prepared using the following primers: B280-B290 / L426 for randomizing bases 1-10 of the target and B269-B278 / L426 for randomizing bases 10-20. The cat gene and srtA upstream region to be assembled with the first and last 10 PCR products were amplified using primers L422 / B268 and L422 / B279, respectively. The assembled constructs were pooled together and transformed with 30 ng in R6 and crR6. After transformation, the cells were plated based on chloramphenicol selection. For each sample, more than 2 × 10⁵ cells were pooled together in 1 ml of THYE, and the genome was analyzed. DNA was extracted using the Promega Wizard kit. The target region was amplified using primers B250 / B251. The PCR product was tagged and run on a single Illumina MiSeq paired-end lane for 300 cycles.

[0209] Analysis of deep sequencing data Randomized PAM: For randomized PAM experiments, 3,429,406 reads were obtained for crR6 and 3,253,998 reads for R6. It is predicted that only half of these will correspond to the PAM target, while the other half will sequence other ends of the PCR product. 1,623,008 crR6 reads and 1,537,131 R6 reads carried error-free target sequences. The possible PAM incidence for each of these reads is shown in the supplementary file. To estimate the functionality of the PAM, its relative ratio in crR6 samples to R6 samples was calculated and expressed as rijklm (where I, j, k, l, and m are one of four possible bases). The following statistical model was constructed: log(rijklm)=μ+b2i+b3j+b4k+b2b3i,j+b3b4j, k+εijklm (In the formula, ε is the residual, b2 is the effect of the second base of PAM, b3 is the effect of the third base, b4 is the effect of the fourth base, b2b3 is the interaction between the second and third bases, and b3b4 is the interaction between the third and fourth bases). Analysis of variance was performed.

[0210] [Table 20]

[0211] When added to this model, b1 or b5 may be considered non-significant, and other interactions can be discarded except for those that can be included. Model selection was performed through a temporal comparison of virtually complete models using the anova method in R. Tukey's honest significance test to determine whether pairwise differences between effects were significant.

[0212] The NGGNN pattern is significantly different from all other patterns and has the strongest effect (see table below).

[0213] To demonstrate that positions 1, 4, or 5 do not influence the NGGNN pattern, the applicants considered only these sequences. These effects are thought to be normally distributed (see QQ plot in Figure 71), and model comparisons using the anova method in R show that the null model is the best, i.e., there is no significant role for b1, b4, and b5.

[0214] [Table 21]

[0215] Partial interference of NAGNN and NNGGN patterns The NAGNN pattern is significantly different from all other patterns, but has considerably less effect than NGGNN (see Tukey's HSD test below).

[0216] Finally, the NTGGN and NCGGN patterns are similar, showing significantly greater CRISPR interference than the NTGHN and NCGHN patterns (where H is A, T, or C), as demonstrated by the Bonferroni-adjusted pairwise Student test.

[0217] [Table 22]

[0218] In summary, these results allow us to conclude that NNGGN patterns generally cause complete interference in the case of NGGGN, or partial interference in the case of NAGGN, NTGGN, or NCGGN.

[0219] [Table 23]

[0220] Randomized target For randomized targeted experiments, 540,726 reads were obtained for crR6 and 753,570 reads for R6. As described above, only half of the reads were predicted to sequence the target end of the PCR product. After filtering out reads carrying targets that were error-free or had a single point mutation, 217,656 and 353,141 reads remained for crR6 and R6, respectively. The relative ratio of each mutant in crR6 samples to R6 samples was calculated (Figure 24c). All mutations outside the seed sequence (13–20 bases away from the PAM) showed complete interference. These sequences were used as reference to determine whether other mutations inside the seed sequence could significantly disrupt the interference. A normal distribution was fitted to these sequences using the fitdistr function of the MASS R package. The 0.99 quantile of the fitted distribution is shown as a dotted line in Figure 24c. Figure 72 shows a histogram of the data density with a fitted normal distribution (black line) and the 0.99th quantile (dotted line).

[0221] [Table 24]

[0222] [Table 25]

[0223] [Table 26]

[0224] [Table 27]

[0225] [Table 28]

[0226] Example 6: Optimization of guide RNA for Streptococcus pyogenes Cas9 (referred to as SpCas9) The applicants improved the RNA in cells by mutating tracrRNA and direct repeat sequences, or by mutating chimeric guide RNA.

[0227] The optimization is based on the observation that thymine stretches (T) were present in the tracrRNA and guide RNA, which can lead to early transcription termination by the pol3 promoter. Therefore, we generated the following optimized sequences. The optimized tracrRNA and the corresponding optimized direct repeat are represented as pairs. Optimized tracrRNA1 (underlined indicates mutation): [ka] Optimized Direct Repeat 1 (Underlined indicates mutation): [ka] Optimized tracrRNA2 (underlined indicates mutation): [ka] Optimized Direct Repeat 2 (Underlined indicates mutation): [ka] The applicants also optimized the chimeric guide RNA for optimal activity in eukaryotic cells. Original guide RNA: [ka] Optimized Chimeric Guide RNA Sequence 1: [ka] Optimized Chimeric Guide RNA Sequence 2: [ka] Optimized Chimeric Guide RNA Sequence 3: [ka]

[0228] The applicants demonstrated that the optimized chimeric guide RNA functions better, as shown in Figure 3. This experiment was performed by simultaneously translocating 293FT cells using Cas9 and U6 guide RNA DNA cassettes to express one of the four RNA forms described above. The guide RNA targets the same target site in the human Emx1 gene locus: "GTCACCTCCAATGACTAGGG".

[0229] Example 7: Optimization of Streptococcus thermophilus LMD-9 CRISPR1 Cas9 (referred to as St1Cas9) The applicants designed the guide chimeric RNA shown in Figure 4.

[0230] St1Cas9 guide RNA can undergo the same type of optimization with respect to SpCas9 guide RNA by degrading the polythymine stretch (T).

[0231] Example 8: Cas9 diversity and mutation The CRISPR-Cas system is an adaptive immune mechanism against invading exogenous DNA used by a diverse range of species, from bacteria to archaea. The type II CRISPR-Cas9 system consists of a set of genes encoding proteins responsible for the "acquisition" of foreign DNA into the CRISPR locus, and a set of genes encoding the "execution" of the DNA cleavage mechanism; these include a DNA nuclease (Cas9), a non-coding transactivating crRNA (tracrRNA), and an array of foreign DNA-derived spacers (crRNA) flanked by direct repeats. During maturation by Cas9, the tracRNA and crRNA double strands guide the Cas9 nuclease to a target DNA sequence defined by the spacer guide sequence, mediating double-strand breaks of DNA near short sequence motifs in the target DNA that are required for cleavage and are specific to each CRISPR-Cas system. Type II CRISPR-Cas systems are found throughout the bacterial kingdom, exhibiting a high degree of diversity in Cas9 protein sequences and sizes, tracrRNA and crRNA direct repeat sequences, genomic composition of their elements, and motif requirements for targeted cleavage. Some species may possess multiple distinct CRISPR-Cas systems.

[0232] The applicants evaluated 207 putative Cas9s from bacterial species identified based on sequence homology with known Cas9s and known subdomains, e.g., the HNH endonuclease domain and the RuvC endonuclease domain [information from Eugene Koonin and Kira Makarova], as well as orthologous structure. Phylogenetic analysis based on protein sequence conservation of this set revealed five families of Cas9, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) (Figures 39 and 40A-F).

[0233] In this embodiment, the applicants demonstrate that the following mutations can convert SpCas9 into a nick-forming enzyme: D10A, E762A, H840A, N854A, N863A, and D986A.

[0234] The applicants provide sequences showing the localization of mutation sites within the SpCas9 gene (Figure 41). The applicants also demonstrate that nickase can still mediate homologous recombination (assay shown in Figure 2). Furthermore, the applicants show that SpCas9 with these mutations does not induce double-strand breaks (individually) (Figure 47).

[0235] Example 9: Supplementary information on the DNA target specificity of RNA-guided Cas9 nucleases Cell culture and translocation Human embryonic kidney (HEK) cell line 293FT (Life Technologies) was maintained at 37°C under 5% CO2 incubation in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0236] 293FT cells were seeded 24 hours prior to translocation on 6-well, 24-well, or 96-well (Corning) plates. Cells were translocated at 80–90% confluence using Lipofectamine 2000 (Life Technologies) according to the manufacturer's recommended protocol. A total of 1 ug of Cas9+ sgRNA plasmid was used for each well in 6-well plates. Unless otherwise specified, a total of 500 ng of Cas9+ sgRNA plasmid was used for each well in 24-well plates. For each well in 96-well plates, 65 ng of Cas9 plasmid was used in a 1:1 molar ratio with the U6-sgRNA PCR product.

[0237] Human embryonic stem cell line HUES9 (Harvard Stem Cell Institute core) was maintained under feeder-free conditions on GelTrex (Life Technologies) in mTesR medium (Stemcell Technologies) supplemented with 100 ug / ml Normocin (InvivoGen). HUES9 cells were transfused using the Amaxa P3 Primary Cell 4-D Nucleofector Kit (Lonza) according to the manufacturer's protocol.

[0238] SURVEYOR nuclease assay for genome modification 293FT cells were transfused using plasmid DNA as described above. The cells were incubated at 37°C for 72 hours after transfusion, and then genomic DNA was extracted. Genomic DNA was extracted using QuickExtract DNA Extraction Solution (Epicentre) according to the manufacturer's protocol. In short, pelletized cells were suspended in QuickExtract solution and incubated at 65°C for 15 minutes and at 98°C for 10 minutes.

[0239] Genomic regions flanking the CRISPR target sites for each gene were PCR-amplified (primers listed in Tables J and K), and the products were purified using QiaQuick Spin Column (Qiagen) according to the manufacturer's protocol. A total of 400 ng of purified PCR product was mixed with 2 μl of 10× Taq DNA Polymerase PCR buffer (Enzymatics), diluted with ultrapure water to a final volume of 20 μl, and subjected to a re-nealing process to enable heteroduplex formation: 95°C for 10 minutes, 95°C to 85°C with a gradient of -2°C / sec, 85°C to 25°C with a gradient of -0.25°C / sec, and maintained at 25°C for 1 minute. After re-nealing, the products were treated with SURVEYOR nuclease and SURVEYOR enhancer S (Transgenomics) according to the manufacturer's recommended protocol and analyzed on 4–20% Novex TBE polyacrylamide gel (Life Technologies). The gels were stained with SYBR Gold DNA stain (Life Technologies) for 30 minutes and imaged using the Gel Doc gel imaging system (Bio-rad). Quantification was based on relative band intensity.

[0240] Northern blot analysis of tracrRNA expression in human cells Northern blotting was performed as described above. Briefly, RNA was heated to 95°C for 5 minutes and then loaded onto an 8% denatured polyacrylamide gel (SequaGel, National Diagnostics). The RNA was then transferred to a pre-hybridized Hybond N+ membrane (GE Healthcare) and crosslinked using a Stratagene UV Crosslinker (Stratagene). The probe was labeled with [gamma-32P]ATP (Perkin Elmer) using T4 polynucleotide kinase (New England Biolabs). After washing, the membrane was exposed to a fluorescent screen for 1 hour and scanned with a phosphorimager (Typhoon).

[0241] Bisulfite sequencing for evaluating DNA methylation status HEK293FT cells were transfused using Cas9 as described above. Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen), and bisulfite was converted using the EZ DNA Methylation-Lightning Kit (Zymo Research). Bisulfite PCR was performed using KAPA2G Robust HotStart DNA Polymerase (KAPA Biosystems) with primers designed using Bisulfite Primer Seeker (Zymo Research, Tables J and K). The resulting PCR amplicons were gel-purified, digested with EcoRI and HindIII, and ligated into a pUC19 backbone before transformation. Subsequently, individual clones were Sanger-sequenced to evaluate the DNA methylation status.

[0242] In vitro transcription and cleavage assays HEK293FT cells were transfused using Cas9 as described above. Whole cell lysates were then prepared using a lysis buffer supplemented with Protease Inhibitor Cocktail (Roche) (20 mM HEPES, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 0.1% Triton X-100). Custom oligonucleotides (Example 10) and the HiScribe T7 In Vitro Transcription Kit (NEB) were used to transcribe T7-driven sgRNA in vitro according to the manufacturer's recommended protocol. To prepare methylation target sites, the pUC19 plasmid was methylated with M.SssI and then linearized with NheI. An in vitro cleavage assay was performed as follows: 20 μL of cleavage reaction material was incubated with 10 μL of cell lysates in 2 μL of cleavage buffer (100 mM HEPES, 500 mM KCl, 25 mM MgCl2, 5 mM DTT, 25% glycerol), in vitro transcribed RNA, and 300 ng of pUC19 plasmid DNA.

[0243] Deep sequencing for evaluating targeting specificity HEK293FT cells plated in a 96-well plate were transfused for 72 hours using Cas9 plasmid DNA and a single guide RNA (sgRNA) PCR cassette, after which genomic DNA was extracted (Figure 72). Genomic regions flanking CRISPR target sites for each gene were amplified by fusion PCR and ionized. The ina P5 adapter and a unique sample-specific barcode were attached to the target amplicon (schematic diagram shown in Figure 73) (Figures 74 and 80 (Example 10)). The PCR product was purified using EconoSpin 96-well Filter Plates (Epoch Life Sciences) according to the manufacturer's recommended protocol.

[0244] Barcoded and purified DNA samples were quantified using the Quant-iT PicoGreen dsDNA Assay Kit or Qubit 2.0 Fluorometer (Life Technologies) and pooled in equimolar ratios. The sequencing library was then deep-sequenced using an Illumina MiSeq Personal Sequencer (Life Technologies).

[0245] Sequencing data analysis and indel detection MiSeq reads were filtered by requiring at least 23 mean Phred quality (Q score) and complete sequence matching with barcode and amplicon forward primers. Reads from on- and off-target loci were first analyzed by performing Smith-Waterman alignment on amplicon sequences containing 50 nucleotides (120 bp total) upstream and downstream of the target site. The alignment was also analyzed for indels from 5 nucleotides upstream to 5 nucleotides downstream of the target site (30 bp total). Target regions analyzed were discarded if any portion of these alignments fell outside the MiSeq read itself, or if the matched base pairs contained less than 85% of their total length.

[0246] For each sample, a negative control provided a metric for the inclusion or exclusion of indels as estimated truncation events. For each sample, indels were counted only if their quality score exceeded μ-σ (where μ is the mean quality score of the negative control corresponding to the sample, and σ is its standard deviation). This resulted in the total target region indel ratio for both the negative controls and their corresponding samples. Using the error ratio q per target region per read of the negative control, the observed indel count n of the sample, and its read count R, the maximum likelihood estimator p for the rate of reads with target regions having true indels was obtained by applying a binomial error model as follows.

[0247] Let E be the (unknown) number of reads in a sample having a target region that was inaccurately counted as having at least one indel.

number

number

[0248] If we assume that all values ​​of the frequency of a target region having a true indel p are equally likely to occur, then Prob(n|p)∝Prob(p|n). Therefore, the maximum likelihood estimator (MLE) for the frequency of a target region having a true indel was set as the value of p that maximizes Prob(n|p). This was then numerically evaluated.

[0249] To place the error bounds at the true indel read frequencies within the sequencing library itself, Wilson score intervals (2) were calculated for each sample to obtain MLE estimates for the true indel target region Rp and the number of reads R. Specifically, the lower limit l and upper limit μ are given by the following equations.

number

[0250] qRT-PCR analysis of relative Cas9 and sgRNA expression 293FT cells plated in 24-well plates were transfused as described above. 72 hours after transfusion, total RNA was recovered using the miRNeasy Micro Kit (Qiagen). Reverse strand synthesis of sgRNA was performed using the qScript Flex cDNA Kit (VWR) and custom first-strand synthesis primers (Table J and K). qPCR analysis was performed using Fast SYBR Green Master Mix (Life Technologies) and custom primers (Table J and K), with GAPDH used as an endogenous control. Relative quantification was calculated using the ΔΔCT method.

[0251] Table I | Target site sequences. Test target sites and required PAMs for the CRISPR system of Streptococcus pyogenes type II. Cells were transfused with Cas9 and either crRNA-tracrRNA or chimeric sgRNA for each target.

[0252] [Table 29]

[0253] [Table 30]

[0254] [Table 31]

[0255] [Table 32]

[0256] Table K|sgRNA architecture sequences. Unless otherwise specified, primers hybridize to the reverse strand of the U6 promoter. The U6 priming site is italicized, guide sequences are shown as N stretches, direct repeat sequences are highlighted in bold, and tracrRNA sequences are underlined. The secondary structures of each sgRNA architecture are shown in Figure 43.

[0257] [Table 33]

[0258] Table L|Cas9 target sites and surrogate PAMs for testing PAM specificity. All target sites for PAM specificity testing are found within the human EMX1 locus.

[0259] [Table 34]

[0260] Example 10: Supplementary arrangement All sequences are oriented from 5' to 3'. For U6 transcription, the underlined T strings act as transcription terminators. >U6-short tracrRNA (Streptococcus pyogenes SF370) [ka] >U6-DR-guide sequence-DR(Streptococcus pyogenes SF370) [ka] sgRNA containing +48 tracrRNA (Streptococcus pyogenes SF370) [ka] sgRNA containing +54 tracrRNA (Streptococcus pyogenes SF370) [ka] sgRNA containing +67tracrRNA (Streptococcus pyogenes SF370) [ka] sgRNA containing +85 tracrRNA (Streptococcus pyogenes SF370) [ka] >CBh-NLS-SpCas9-NLS [ka] [ka] [ka] [ka] >Amplicon Sequencing for EMX1 Guides 1.1, 1.14, and 1.17 [ka] >Amplicon Sequencing for EMX1 Guide 1.2, 1.16 [ka] >Amplicon Sequencing for EMX1 Guides 1.3, 1.13, and 1.15 [ka] >Amplicon Sequencing for EMX1 Guide 1.6 [ka] >Amplicon Sequencing for EMX1 Guide 1.10 [ka] >Amplicon Sequencing for EMX1 Guide 1.11, 1.12 [ka] >Amplicon Sequencing for EMX1 Guide 1.18, 1.19 [ka] >Amplicon Sequencing for EMX1 Guide 1.20 [ka] T7 promoter F primer for annealing with the target chain [ka] > Oligosaccharide containing pUC19 target site 1 for methylation (T7 reverse) [ka] > Oligosaccharide containing pUC19 target site 2 for methylation (T7 reverse) [ka]

[0261] Example 11: Oligo-mediated Cas9-induced homologous recombination Oligo-homologous recombination testing is a comparison of efficiency across different Cas9 variants and different HR templates (oligo vs. plasmid).

[0262] 293FT cells were used. SpCas9 = wild-type Cas9, and SpCas9n = nickase Cas9 (D10A). The chimeric RNA target was EMX1 protospacer target 1, the same as in Examples 5, 9, and 10, and the oligonucleotide was synthesized by IDT using PAGE purification.

[0263] Figure 44 shows the design of the oligo DNA used as the homologous recombination (HR) template in this experiment. The long-chain oligo contains 100 bp homology to the EMX1 locus and the HindIII restriction site. 293FT cells were simultaneously transfused, firstly, with a plasmid containing chimeric RNA and wild-type cas9 protein targeting the human EMX1 locus, and secondly, with the oligo DNA as the HR template. The sample was from 293FT cells collected 96 hours after transfusion with Lipofectamine 2000. All products were amplified using EMX1HR primers, gel-purified, and then digested with HindIII to detect the efficiency of HR template integration into the human genome.

[0264] Figures 45 and 46 show a comparison of HR efficiencies induced by different combinations of Cas9 protein and HR templates. The Cas9 construct used was either wild-type Cas9 or the nickase version of Cas9 (Cas9n). The HR templates used were antisense oligo DNA (antisense oligo in the upper figure), sense oligo DNA (sense oligo in the upper figure), or plasmid HR templates (HR templates in the upper figure). The sense / antisense definition is that the sense strand of the genome is defined as the actively transcribed strand having a sequence corresponding to the mRNA being transcribed. HR efficiency is shown as the percentage of HindIII digested bands relative to all genomic PCR amplification products (numbers below).

[0265] Example 12: Autistic Mice Recent large-scale sequencing initiatives have yielded numerous disease-associated genes. Gene discovery is only the beginning of understanding what that gene is and how it leads to disease phenotypes. Current techniques and approaches for studying candidate genes are slow and cumbersome. Gene targeting and gene knockout, typical criteria, require considerable investment of time and resources, both financially and in terms of research personnel. We have utilized the hSpCas9 nuclease to target many target genes and have configured it to do so with high efficiency and low turnaround compared to any other technique. Due to the high efficiency of hSpCas9, we can perform RNA injection in mouse zygotes and immediately obtain genome-modified animals without the need for any preliminary gene targeting in mESCs.

[0266] Chromodomain helicase DNA-binding protein 8 (CHD8) is a critically important gene involved in early vertebrate development and morphogenesis. Mice lacking CHD8 die during embryonic development. Mutations in the CHD8 gene are associated with autism spectrum disorder in humans. This association was established in three different papers published simultaneously in Nature. The three identical studies identified a large number of genes associated with autism spectrum disorder. Our objective was to create knockout mice for the four genes found in all the papers: Chd8, Katnal2, Kctd13, and Scn2a. In addition, we selected two other genes associated with autism spectrum disorder, schizophrenia, and ADHD: GIT1, CACNA1C, and CACNB2. Finally, we decided to target MeCP2 as a positive control.

[0267] For each gene, the applicants believe there is a high probability of knocking out the gene. Two gRNAs were designed. The knockout was hSpCas9 nuclease cleavage of double strands. Furthermore, error-prone DNA repair pathways are created, non-homologous end joining is performed, cleavage is corrected, and sudden changes occur. It occurs after mutation is created. The most likely outcome is a frame that knocks out the gene. This is a shift mutation. The targeting strategy is to identify a PAM sequence NGG that is present in the genome. This involved finding protospacers in the exons of unusual genes. The protospacer in the first exon, which is harmful, was prioritized.

[0268] Each gRNA was validated by liposome transient simultaneous translocation with hSpCas9 in the mouse cell line Neuro-N2a. 72 hours after translocation, genomic DNA was purified using QuickExtract DNA from Epicentre. PCR was performed to amplify the target locus. Subsequently, the SURVEYOR Mutation Detection Kit from Transgenomics was used. The SURVEYOR results for each gRNA and their respective controls are shown in Figure A1. A positive SURVEYOR result is represented by one large band corresponding to the genomic PCR and two smaller bands, which are the products of the SURVEYOR nuclease that create double-strand breaks at the mutation site. The average cleavage efficiency of each gRNA was also measured for each gRNA. The gRNA selected for injection was the most unique and maximally efficient gRNA within the genome.

[0269] RNA (hSpCas9+gRNA RNA) was injected into the pronucleus of a zygote and then transplanted into a surrogate mother. The surrogate mother was allowed to reach full term, and the offspring was sampled by tail snip 10 days after birth. DNA was extracted and used as a template for PCR, and then processed with SURVEYOR. Furthermore, the PCR products were sent for sequencing. Animals detected as positive in either the SURVEYOR assay or PCR sequencing had their genomic PCR products cloned into a pUC19 vector and sequenced to determine putative mutations from their respective alleles.

[0270] To date, the offspring mice from Chd8 targeting experiments have been fully processed up to the time of allele sequencing. Surveyor results for 38 surviving offspring (lanes 1-38), 1 dead offspring (lane 39), and 1 wild-type offspring for comparison (lane 40) are shown in Figure A2. gRNA Chd8.2 was injected into offspring 1-19, and gRNA Chd8.3 was injected into offspring 20-38. Of the 38 surviving offspring, 13 were positive for mutations. The one dead offspring also had mutations. No mutations were detected in the wild-type sample. Genomic PCR sequencing was consistent with the findings of the SURVEYOR assay.

[0271] Example 13: CRISPR / Cas-mediated transcription modulation Figure 67 shows the design of a CRISPR-TF (transcription factor) with transcriptional activation activity. A chimeric RNA is expressed via the U6 promoter, while a human codon-optimized double mutant version of the Cas9 protein (hSpCas9m), operably bound to three NLS and VP64 functional domains, is expressed via the EF1a promoter. The double mutants D10A and H840A render the Cas9 protein unable to induce any cleavage, but its ability to bind to target DNA when guided by the chimeric RNA was maintained.

[0272] Figure 68 shows transcriptional activation of the human SOX2 gene by the CRISPR-TF system (chimeric RNA and Cas9-NLS-VP64 fusion protein). 293FT cells were transfused with a plasmid carrying two components: (1) a different chimeric RNA driven by U6 targeting a 20 bp sequence within or around the human SOX2 genomic locus, and (2) an hSpCas9m (double mutant)-NLS-VP64 fusion protein driven by EF1a. 96 hours after transfusion, 293FT cells were harvested, and the level of activation was measured by introducing mRNA expression using a qRT-PCR assay. All expression levels were normalized against a control group (gray bars) representing results from cells transfused with a CRISPR-TF backbone plasmid without chimeric RNA. The qRT-PCR probe used to detect SOX2 mRNA was the Taqman Human Gene Expression Assay (Life Technologies). All experiments represent data from three biological replicas, n=3, and the error bars indicate the standard error (SEM).

[0273] Example 14: NLS: Cas9NLS 293FT cells were transfused using a plasmid containing two components: (1) an EF1a promoter driving the expression of Cas9 with a different NLS design (wild-type human codon-optimized SpCas9), and (2) a U6 promoter driving identical chimeric RNA targeting the human EMX1 locus.

[0274] Cells were harvested 72 hours after translocation and then extracted using 50 μl of QuickExtract genomic DNA extraction solution according to the manufacturer's protocol. Target EMX1 genomic DNA was amplified by PCR and then gel-purified on a 1% agarose gel. Genomic PCR products were re-annealed and subjected to Surveyor assays according to the manufacturer's protocol. Genomic cleavage efficiency of different constructs was measured using SDS-PAGE on 4–12% TBE-PAGE gels (Life Technologies), analyzed and quantified using ImageLab (Bio-rad) software, all according to the manufacturer's protocol.

[0275] Figure 69 shows the designs of different Cas9 NLS constructs. All Cas9s were human codon-optimized versions of SpCas9. The NLS sequence was ligated to the cas9 gene either at the N-terminus or C-terminus. All Cas9 variants with different NLS designs were cloned into a skeletal vector containing them so that they were driven by the EF1a promoter. A chimeric RNA targeting the human EMX1 locus, driven by the U6 promoter, was present on the same vector, forming a two-component system together.

[0276] Table shows the results of M.Cas9 NLS design tests. Quantification of genomic cleavage of different Cas9-NLS constructs by surveyor assay.

[0277] [Table 35]

[0278] Figure 70 shows the efficiency of genome cleavage induced by Cas9 variants carrying different NLS designs. The percentages represent the portion of human EMX1 genomic DNA cleaved by each construct. All experiments represent data from three biological replicas, n=3, and error bars indicate standard error (SEM).

[0279] Example 15: Engineering of microalgae using Cas9 How to deliver Cas9

[0280] Method 1: The applicants deliver Cas9 and guide RNA using a vector that expresses Cas9 under the control of a constitutive promoter, e.g., Hsp70A-Rbc S2 or beta-2-tubulin.

[0281] Method 2: The applicants deliver Cas9 and T7 polymerases using a vector expressing Cas9 and T7 polymerases under the control of a constitutive promoter, e.g., Hsp70A-Rbc S2 or beta-2-tubulin. Guide RNA is used as guide R The NA is delivered using a vector containing a T7 promoter that drives the NA.

[0282] Method 3: The applicants deliver Cas9 mRNA and in vitro transcribed guide RNA to algal cells. The RNA can be transcribed in vitro. The Cas9 mRNA consists of a coding region for Cas9 and a 3'UTR from Cop1 to ensure stabilization of the Cas9 mRNA.

[0283] For homologous recombination, the applicants provide additional homologous recombination repair templates.

[0284] Sequences for the cassette that drives Cas9 expression under the control of the beta-2 tubulin promoter, followed by the 3'UTR of Cop1. [ka] [ka] [ka] [ka]

[0285] Sequences for the cassette that drives T7 polymerase expression under the control of the beta-2 tubulin promoter, followed by the 3'UTR of Cop1: [ka] [ka]

[0286] The sequence of the guide RNA driven by the T7 promoter (T7 promoter, N represents the targeting sequence): [ka]

[0287] Gene delivery: Chlamydomonas reinhardtii strains CC-124 and CC-125 from the Chlamydomonas Resource Center will be used for electroporation. The electroporation protocol will follow the standard recommended protocol from the GeneArt Chlamydomonas Engineering kit.

[0288] Furthermore, the applicants generate a lineage of Chlamydomonas reinhardtii that constitutively expresses Cas9. This can be achieved by using pChlamy1 (linearized using PvuI) and selecting hygromycin-resistant colonies. The sequence for pChlamy1 containing Cas9 is shown below. In this method to achieve gene knockout, only the delivery of RNA for the guide RNA is required. For homologous recombination, the applicants deliver the guide RNA and a linearized homologous recombination template. pChlamy1-Cas9: [ka] [ka] [ka] [ka] [ka] [ka]

[0289] For all modified Chlamydomonas reinhardtii cells, the applicants confirmed good modification using PCR, SURVEYOR nuclease assay, and DNA sequencing.

[0290] Example 16: Use of Cas9 as a transcriptional repressor in bacteria The ability to artificially control transcription is essential for both the study of gene function and the construction of synthetic gene networks with desired properties. The applicants hereby describe the use of the RNA-guided Cas9 protein as a programmable transcriptional repressor.

[0291] The applicants have already demonstrated how the Cas9 protein from Streptococcus pyogenes SF370 can be used to target genome editing in Streptococcus pneumoniae. In this study, the applicants engineered a minimal CRISPR system containing cas9, tracrRNA, and repeats, called the crR6Rk strain. The D10A-H840 mutation was introduced into the cas9 in this strain to obtain the crR6Rk** strain. Four spacers targeting different positions in the bgaA β-galactosidase gene promoter were cloned in a CRISPR array supported by the previously described pDB98 plasmid. The applicants observed an X to Y-fold reduction in β-galactosidase activity depending on the targeted position, demonstrating the potential of Cas9 as a programmable repressor (Figure 73).

[0292] To achieve Cas9** repression in Escherichia coli, a green fluorescent protein (GFP) reporter plasmid (pDB127) was constructed to express the gfpmut2 gene from a constitutive promoter. The promoter was designed to support several NPP PAMs on both strands to measure the effect of Cas9** binding at various positions. The applicant introduced the D10A-H840 mutation into pCas9 of the described plasmid, which supports a minimal CRISPR array designed for easy cloning of tracrRNA, cas9, and novel spacers. Twenty-two different spacers were designed to target different regions of the gfpmut2 promoter and open reading frame. Approximately 20-fold reduction in fluorescence was observed when targeting the -35 and -10 promoter elements and regions overlapping with or adjacent to the Shine-Dalgano sequence. Targets on both strands showed similar levels of repression. These results suggest that binding of Cas9** to any position in the promoter region interferes with transcription initiation through putative steric inhibition of RNAP binding.

[0293] To determine whether Cas9** can interfere with transcriptional elongation, we directed it to the reading frame of gpfmut2. Reduction in fluorescence was observed in both targeted coding and non-coding strands, suggesting that Cas9 binding is indeed potent enough to represent impairment to the RNAP on which it acts. However, a 40% reduction in expression was observed when the coding strand was targeted, compared to a 20-fold reduction in the non-coding strand (Figure 21b, comparing T9, T10, and T11 with B9, B10, and B11). To directly determine the effect of Cas9** binding on transcription, we extracted RNA from strains carrying either T5, T10, B10, or a control construct that did not target pDB127, and subjected it to Northern blotting analysis using either a probe that binds before (B477) or after (B510) the B10 and T10 target sites. Consistent with our fluorescence method, gfpmut2 transcription was not detected when Cas9** was targeted to the promoter region (T5 target), and transcription was observed after targeting the T10 region. Interestingly, smaller transcripts were observed for the B477 probe. This band corresponds to the predicted size of transcripts interrupted by Cas9** and is a direct indicator of transcription termination caused by dgRNA::Cas9** binding to the coding strand. Surprisingly, we did not detect any transcripts when targeting the non-coding strand (B10). This result suggests that the mRNA was degraded, as Cas9** binding to the B10 region is unlikely to interfere with transcription initiation. DgRNA::Cas9 has been shown to bind to ssRNA in vitro. We speculate that binding may trigger mRNA degradation by host nucleases. Indeed, ribosome stalling can induce cleavage of translatable mRNA in E. coli (E. coli).

[0294] Some applications require precise regulation of gene expression rather than complete suppression. The applicant sought to achieve intermediate levels of suppression through the introduction of mismatches that weaken crRNA / target interactions. The applicant created a series of spacers based on B1, T5, and B10 constructs with an increased number of mutations in the 5' end of the crRNA. Up to eight mutations in B1 and T5 did not affect the suppression level, and a gradual increase in fluorescence was observed for additional mutations.

[0295] The repression observed only for 8nt matches between crRNA and its target raises the issue of off-targeting effects of using Cas9** as a transcription regulator. Since a good PAM (NGG) is also required for Cas9 binding, the number of nucleotides to match to obtain the same level of respiration is 10. 10nt matches occur randomly about once every 1 Mbp, and therefore such sites are likely to be found even in small bacterial genomes. However, for effective repression of transcription, such sites must be located within the promoter region of the gene, which significantly reduces the likelihood of off-targeting. We have also shown that gene expression can be affected when the non-coding strand of the gene is targeted. For this to occur, the random target must be located to the right, but such events are relatively likely to occur. In fact, during the course of this study, we were unable to construct one of the spacers designed on pCas9**. We later found that this spacer exhibits a 12bp match adjacent to a good PAM in the essential murC gene. Such off-targeting can be easily avoided by systematically blasting the spacers during the design process.

[0296] Aspects of the present invention are further described in the following numbered paragraphs: 1. A vector system containing one or more vectors, a. A first regulatory element operably bound to one or more insertion sites for inserting a traer mate sequence and a guide sequence upstream of the traer mate sequence (the guide sequence, when expressed, directs the sequence-specific binding of the CRISPR complex to a target sequence in eukaryotic cells, and the CRISPR complex comprises a CRISPR enzyme that complexes with (1) a guide sequence which hybridizes to the target sequence, and (2) a traer mate sequence which hybridizes to the traer sequence); and b. A second regulatory element operably bound to the enzyme coding sequence encoding the CRISPR enzyme, which includes a nuclear localization sequence. Includes; Components (a) and (b) are located on the same or different vectors of the system. Vector-based.

[0297] 2. The vector system described in paragraph 1, wherein component (a) further comprises a traer sequence downstream of the traer mate sequence under the control of a first regulatory element.

[0298] 3. The vector system described in paragraph 1, further comprising two or more guide sequences operably bound to a first regulatory element, each of which, when expressed, directs the CRISPR complex to sequence-specific binding to different target sequences in eukaryotic cells.

[0299] 4. The vector system described in paragraph 1, comprising the traer sequence under the control of a third regulatory element.

[0300] 5. The vector system described in paragraph 1, wherein the traer sequence exhibits at least 50% sequence complementarity along the length of the traer mate sequence when optimally aligned.

[0301] 6. The vector system according to paragraph 1, wherein the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive the accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.

[0302] 7. The vector system described in paragraph 1, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

[0303] 8. The vector system described in paragraph 1, in which the CRISPR enzyme is the Cas9 enzyme.

[0304] 9. The vector system described in paragraph 1, in which the CRISPR enzyme is codon-optimized for expression in eukaryotic cells.

[0305] 10. The vector system described in paragraph 1, wherein the CRISPR enzyme is directed to cleave one or two strands at the localization of the target sequence.

[0306] 11. The vector system described in paragraph 1, wherein the CRISPR enzyme lacks DNA strand cleavage activity.

[0307] 12. The vector system described in paragraph 1, wherein the first regulatory element is a polymerase III promoter.

[0308] 13. The vector system described in paragraph 1, wherein the second regulatory element is a polymerase II promoter.

[0309] 14. The vector system described in paragraph 4, wherein the third regulatory element is a polymerase III promoter.

[0310] 15. The vector system described in paragraph 1, wherein the guide sequence is at least 15 nucleotides long.

[0311] 16. The vector system described in paragraph 1, wherein less than 50% of the nucleotides of the guide sequence are involved in self-complementary base pairing when optimally folded.

[0312] 17. A vector comprising a regulatory element operably bound to an enzyme coding sequence encoding a CRISPR enzyme containing one or more nuclear localization sequences, wherein the regulatory element drives the transcription of the CRISPR enzyme in a eukaryotic cell to accumulate in a detectable amount in the nucleus of the eukaryotic cell.

[0313] 18. The vector system according to paragraph 17, wherein the regulatory element is a polymerase II promoter.

[0314] 19. The vector system described in paragraph 17, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

[0315] 20. The vector system described in paragraph 17, wherein the CRISPR enzyme is a Cas9 enzyme.

[0316] 21. The vector system according to paragraph 17, wherein the CRISPR enzyme lacks the ability to cleave one or more strands of the target sequence to which it binds.

[0317] 22. A CRISPR enzyme containing one or more nuclear localization sequences of sufficient strength to drive the accumulation of a detectable amount of CRISPR enzyme in the nucleus of a eukaryotic cell.

[0318] 23. A type II CRISPR system enzyme, as described in paragraph 22.

[0319] 24. The Cas9 enzyme, which is the CRISPR enzyme described in paragraph 22.

[0320] 25. A CRISPR enzyme described in paragraph 22 that lacks the ability to cleave one or more strands of the target sequence to which it binds.

[0321] 26.a. A first regulatory element operably bound to one or more insertion sites for inserting a traer mate sequence and a guide sequence upstream of the traer mate sequence (the guide sequence, if expressed, directs the sequence-specific binding of the CRISPR complex to a target sequence in eukaryotic cells, and the CRISPR complex comprises a CRISPR enzyme that complexes with (1) a guide sequence which hybridizes to the target sequence, and (2) a traer mate sequence which hybridizes to the traer sequence); and / or b. A second regulatory element operably bound to the enzyme coding sequence encoding the CRISPR enzyme, which includes a nuclear localization sequence. Eukaryotic host cells that include this cell.

[0322] 27. A eukaryotic host cell as described in paragraph 26, comprising components (a) and (b).

[0323] 28. A eukaryotic host cell as described in paragraph 26, wherein component (a), component (b), or components (a) and (b) are stably integrated into the genome of the host eukaryotic cell.

[0324] 29. A eukaryotic host cell as described in paragraph 26, wherein component (a) further comprises a traer sequence downstream of the traer mate sequence under the control of a first regulatory element.

[0325] 30. A eukaryotic host cell as described in paragraph 26, further comprising two or more guide sequences operably bound to a first regulatory element, each of which, when expressed, directs the CRISPR complex to sequence-specific binding to different target sequences in the eukaryotic cell.

[0326] 31. A eukaryotic host cell according to paragraph 26, further comprising a third regulatory element operably bound to the traer sequence.

[0327] 32. Eukaryotic host cells as described in paragraph 26, wherein the traer sequence exhibits at least 50% sequence complementarity along the length of the traer mate sequence when optimally aligned.

[0328] 33. A eukaryotic host cell as described in paragraph 26, comprising one or more nuclear localization sequences of sufficient strength to drive the accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.

[0329] 34. Eukaryotic host cells as described in paragraph 26, in which the CRISPR enzyme is a type II CRISPR system enzyme.

[0330] 35. Eukaryotic host cells as described in paragraph 26, where the CRISPR enzyme is the Cas9 enzyme.

[0331] 36. Eukaryotic host cells as described in paragraph 26, in which the CRISPR enzyme has been codon-optimized for expression in eukaryotic cells.

[0332] 37. Eukaryotic host cells as described in paragraph 26, in which the CRISPR enzyme is directed to cleave one or two strands at the localization of a target sequence.

[0333] 38. Eukaryotic host cells, as described in paragraph 26, in which the CRISPR enzyme lacks DNA strand cleavage activity.

[0334] 39. Eukaryotic host cells as described in paragraph 26, wherein the first regulatory element is the polymerase III promoter.

[0335] 40. Eukaryotic host cells as described in paragraph 26, in which the second regulatory element is the polymerase II promoter.

[0336] 41. Eukaryotic host cells as described in paragraph 31, wherein the third regulatory element is the polymerase III promoter.

[0337] 42. Eukaryotic host cells as described in paragraph 26, wherein the guide sequence is at least 15 nucleotides long.

[0338] 43. Eukaryotic host cells as described in paragraph 26, in which less than 50% of the nucleotides of the guide sequence are involved in self-complementary base pairing when optimally folded.

[0339] 44. A non-human animal containing a eukaryotic host cell as described in any one of paragraphs 26-43.

[0340] 45. A kit including instructions for use of the vector system and the kit, wherein the vector system is a. A first regulatory element operably bound to one or more insertion sites for inserting a traer mate sequence and a guide sequence upstream of the traer mate sequence (the guide sequence, when expressed, directs the sequence-specific binding of the CRISPR complex to a target sequence in eukaryotic cells, and the CRISPR complex comprises a CRISPR enzyme that complexes with (1) a guide sequence which hybridizes to the target sequence, and (2) a traer mate sequence which hybridizes to the traer sequence); and / or b. A second regulatory element operably bound to the enzyme coding sequence encoding the CRISPR enzyme, which includes a nuclear localization sequence. A kit that includes this.

[0341] 46. ​​The kit described in paragraph 45, comprising components (a) and (b) located on the same or different vectors of the system.

[0342] 47. The kit described in paragraph 45, wherein component (a) further comprises a traer sequence downstream of a traer mate sequence under the control of a first regulatory element.

[0343] 48. The kit according to paragraph 45, wherein component (a) further comprises two or more guide sequences operably bound to a first regulatory element, each of which, when expressed, directs the CRISPR complex to sequence-specific binding to different target sequences in eukaryotic cells.

[0344] 49. The kit described in paragraph 45, wherein the system includes a traer sequence under the control of a third regulatory element.

[0345] 50. The kit described in paragraph 45, wherein the traer sequence exhibits at least 50% sequence complementarity along the length of the traer mate sequence when optimally aligned.

[0346] 51. The kit according to paragraph 45, wherein the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive the accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.

[0347] 52. The kit described in paragraph 45, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

[0348] 53. The kit described in paragraph 45, in which the CRISPR enzyme is the Cas9 enzyme.

[0349] 54. The kit described in paragraph 45, in which the CRISPR enzyme has been codon-optimized for expression in eukaryotic cells.

[0350] 55. The kit described in paragraph 45, wherein the CRISPR enzyme is directed to cleave one or two strands at the localization of a target sequence.

[0351] 56. The kit described in paragraph 45, in which the CRISPR enzyme lacks DNA strand cleavage activity.

[0352] 57. The kit described in paragraph 45, wherein the first regulatory element is a polymerase III promoter.

[0353] 58. The kit described in paragraph 45, wherein the second regulatory element is a polymerase II promoter.

[0354] 59. The kit described in paragraph 49, wherein the third regulatory element is a polymerase III promoter.

[0355] 60. The kit described in paragraph 45, wherein the guide sequence is at least 15 nucleotides long.

[0356] 61. The kit described in paragraph 45, wherein less than 50% of the nucleotides of the guide sequence are involved in self-complementary base pairing when optimally folded.

[0357] 62. A computer system for selecting candidate target sequences within nucleic acid sequences in eukaryotic cells for targeting by the CRISPR complex, a. A memory device configured to receive and / or store the nucleic acid sequence; and b. One or more processors, either alone or in combination, programmed to (i) localize a CRISPR motif sequence within the nucleic acid sequence, and (ii) select sequences adjacent to the localized CRISPR motif sequence as candidate target sequences to which the CRISPR complex binds. A system that includes this.

[0358] 63. The computer system according to paragraph 62, wherein the localization step includes identifying a CRISPR motif sequence localized less than approximately 500 nucleotides away from the target sequence.

[0359] 64. The computer system described in paragraph 62, wherein the candidate target sequence is at least 10 nucleotides long.

[0360] 65. The computer system described in paragraph 62, wherein the nucleotide at the 3' end of the candidate target sequence is localized to approximately 10 nucleotides or less upstream of the CRISPR motif sequence.

[0361] 66. The computer system described in paragraph 62, wherein the nucleic acid sequence in the eukaryotic cell is endogenous with respect to the eukaryotic genome.

[0362] 67. The nucleic acid sequence in the eukaryotic cell is exogenous with respect to the eukaryotic genome, as described in claim 62. The computer system.

[0363] 68. A computer-readable medium comprising code that implements a method for selecting candidate target sequences within nucleic acid sequences in eukaryotic cells for targeting by a CRISPR complex, wherein the method comprises (a) localizing a CRISPR motif sequence within the nucleic acid sequence, and (b) selecting sequences adjacent to the localized CRISPR motif sequence as candidate target sequences to which the CRISPR complex will bind.

[0364] 69. The computer-readable medium according to paragraph 68, wherein the localization step comprises localizing a CRISPR motif sequence located less than approximately 500 nucleotides away from the target sequence.

[0365] 70. The computer-readable medium according to paragraph 68, wherein the candidate target sequence is at least 10 nucleotides long.

[0366] 71. A computer-readable medium as described in paragraph 68, wherein the nucleotide at the 3' end of the candidate target sequence is localized to approximately 10 nucleotides upstream or less of the CRISPR motif sequence.

[0367] 72. A computer-readable medium, as described in paragraph 68, in which the nucleic acid sequence in the eukaryotic cell is endogenous with respect to the eukaryotic genome.

[0368] 73. A computer-readable medium, as described in paragraph 68, in which the nucleic acid sequence in the eukaryotic cell is exogenous to the eukaryotic genome.

[0369] 74. A method for modifying a target polynucleotide in a eukaryote, comprising binding a CRISPR complex to the target polynucleotide to cause cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme that is complexed with a guide sequence which hybridizes to a target sequence in the target polynucleotide, and the guide sequence is then bound to a traer mate sequence which hybridizes to a traer sequence.

[0370] 75. The method according to paragraph 74, wherein the cleavage comprises cleaving one or two strands at the localization of the target sequence by the CRISPR enzyme.

[0371] 76. The method according to paragraph 74, wherein the cleavage results in a decrease in the transcription of the target gene.

[0372] 77. The method according to paragraph 74, further comprising repairing the cleavage target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair results in a mutation comprising the insertion, deletion, or substitution of one or more nucleotides in the target polynucleotide.

[0373] 78. The method according to paragraph 77, wherein the mutation results in one or more amino acid changes in a protein expressed from a gene containing a target sequence.

[0374] 79. The method according to paragraph 74, further comprising delivering one or more vectors to the eukaryotic cells, wherein one or more vectors drive the expression of one or more CRISPR enzymes, guide sequences bound to a tracer mate sequence, and tracer sequences.

[0375] 80. The method according to paragraph 79, wherein the vector is delivered into a target eukaryotic cell.

[0376] 81. The method according to paragraph 74, wherein the modification is carried out in the eukaryotic cells in a cell culture.

[0377] 82. The method according to paragraph 74, further comprising isolating the eukaryotic cells from the subject before the modification.

[0378] 83. The method according to paragraph 82, further comprising returning the eukaryotic cells and / or cells derived therefrom to the subject.

[0379] 84. A method for modifying the expression of a polynucleotide in a eukaryotic cell, comprising binding a CRISPR complex to a polynucleotide, wherein the binding results in an increase or decrease in the expression of the polynucleotide; the CRISPR complex comprises a CRISPR enzyme complexing with a guide sequence which hybridizes to a target sequence within the target polynucleotide, the guide sequence which then hybridizes to a tracer sequence. The method of binding to the mate sequence.

[0380] 85. The method according to paragraph 74, further comprising delivering one or more vectors to the eukaryotic cells, wherein one or more vectors drive the expression of one or more CRISPR enzymes, guide sequences bound to a tracer mate sequence, and tracer sequences.

[0381] 86. A method for generating model eukaryotic cells containing mutated disease genes, a. Introducing one or more vectors into eukaryotic cells (one or more vectors drive th...

Claims

1. (a) Cas9 protein or polynucleotide encoding Cas9 protein, wherein the Cas9 protein is S. pyogenes Cas9 and is fused with two or more nuclear localization signals (NLS); (b) A CRISPR-Cas9 chimeric RNA comprising NN An engineered CRISPR-Cas9 system comprising the chimeric RNA and the Cas9 protein, wherein the chimeric RNA and the Cas9 protein are capable of forming a CRISPR complex in eukaryotic cells, and the guide sequence is capable of directing sequence-specific binding of the CRISPR complex to a target sequence adjacent to the PAM at a target genomic locus of interest in eukaryotic cells.

2. (a) Cas9 protein or polynucleotide encoding Cas9 protein, wherein the Cas9 protein is S. pyogenes Cas9 and is fused with two or more nuclear localization signals (NLS); (b) A CRISPR-Cas9 chimeric RNA comprising NNNNNNNNNNNNNNNNNNNNNNNNNNGUUUUUAGAGCUAGAAAAUAGCAAGUUAAAAAAAUAGCUAGUUCCGUUAUCAACUUGAAAAGUG, wherein NNNNNNNNNNNNNNNNNNNNNNNN is a guide sequence that can hybridize to a target sequence adjacent to a protospacer facing motif (PAM) at a target genomic locus of interest in a eukaryotic cell, and the chimeric RNA further comprises a poly-U sequence: An engineered CRISPR-Cas9 system comprising the chimeric RNA and the Cas9 protein, wherein the chimeric RNA and the Cas9 protein are capable of forming a CRISPR complex in eukaryotic cells, and the guide sequence is capable of directing sequence-specific binding of the CRISPR complex to a target sequence adjacent to the PAM at a target genomic locus of interest in eukaryotic cells.

3. (a) Cas9 protein or polynucleotide encoding Cas9 protein, wherein the Cas9 protein is S. pyogenes Cas9 and is fused with two or more nuclear localization signals (NLS); (b) A CRISPR-Cas9 chimeric RNA comprising NN An engineered CRISPR-Cas9 system comprising the chimeric RNA and the Cas9 protein, wherein the chimeric RNA and the Cas9 protein are capable of forming a CRISPR complex in eukaryotic cells, and the guide sequence is capable of directing sequence-specific binding of the CRISPR complex to a target sequence adjacent to the PAM at a target genomic locus of interest in eukaryotic cells.

4. An engineered CRISPR-Cas9 system according to any one of claims 1 to 3, wherein the PAM is NGG.

5. An engineered CRISPR-Cas9 system according to any one of claims 1 to 3, wherein the chimeric RNA comprises one or more modified nucleotides.

6. An engineered CRISPR-Cas9 system according to any one of claims 1 to 3, wherein the chimeric RNA comprises one or more methylated nucleotides or nucleotide analogs.

7. Two or more NLSs operate independently, PKKKRKV, KRPAATKKAGQAKKKK, An engineered CRISPR-Cas9 system according to any one of claims 1 to 3, selected from the group consisting of PAAKRVKLD, RQRRNELKRSP, NQSSNFGPMKGGGNFGGGRSSGPYGGGGQYFAKPRNQGGY, RMRIZFKNKGKDTAELRRRRRVEVSVELRKAKKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKKK, and RKCLQAGMNLEARKTKK.

8. The engineered CRISPR-Cas9 system according to claim 7, wherein at least one of the NLSs includes PKKKRKV.

9. An engineered CRISPR-Cas9 system according to any one of claims 1 to 3, wherein the Cas9 protein comprises the D10A, H840A, N854A, or N863A mutation.

10. The engineered CRISPR-Cas9 system according to claim 9, wherein the Cas9 protein is fused with at least one heterologous protein domain.

11. The engineered CRISPR-Cas9 system according to claim 10, wherein the heterologous protein domain is selected from the group consisting of an epitope tag, a reporter sequence, and a protein domain having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, or nucleic acid binding activity.

12. An engineered CRISPR-Cas9 system according to any one of claims 1 to 3, wherein the polynucleotide encoding the Cas9 protein is codon-optimized for expression in eukaryotic cells.

13. An engineered CRISPR-Cas9 system according to any one of claims 1 to 3, wherein the polynucleotide encoding the Cas9 protein includes a polyadenylation signal.

14. An engineered CRISPR-Cas9 system according to any one of claims 1 to 3, wherein the CRISPR-Cas9 system is contained in a liposome for delivery.

15. An engineered CRISPR-Cas9 system according to any one of claims 1 to 3, further comprising an exogenous polynucleotide for targeted incorporation into DNA cleavage introduced by the CRISPR complex.

16. A pharmaceutical composition comprising an engineered CRISPR-Cas9 system according to any one of claims 1 to 3 for use in the treatment of a hereditary disease or disorder, wherein such use does not involve a step of modifying the genetic identity of a human germline.

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