S. pyogenes CAS9 mutant gene and polypeptide encoded thereby

Abstract

This invention provides mutant Cas9 nucleic acids and proteins for use in the CRISPR / Cas endonuclease system, as well as methods for using them. [Solution] The present invention relates to an isolated mutant Cas9 protein that is active in the CRISPR / Cas endonuclease system, wherein the CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system. The present invention also includes an isolated nucleic acid encoding the mutant Cas9 protein, a ribonucleoprotein complex, and a CRSPR / Cas endonuclease system having the mutant Cas9 protein exhibiting reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 405,601, “NOVEL S.PYOGENES CAS9 MUTATIONS THAT REDUCE OFF TARGET GENE EDITING WHILE MAINTAINING ON TARGET POTENCY,” filed on 7 October 2016 under Section 119 of the U.S. Patent Act, the entire contents of which are incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on December 18, 2015, is named IDT01-009-PCT_ST25.txt and is ___ bytes in size.

[0003] Field of Invention This invention relates to Cas9 mutant genes, polypeptides encoded thereby, and their use in CRISPR-Cas system compositions. [Background technology]

[0004] The use of clustered short repeat palindromic sequences (CRISPR) and associated Cas proteins (CRISPR-Cas system) with regular spacing for site-specific DNA cleavage has demonstrated excellent potential for numerous biological applications. CRISPR is used in genome editing, genome-scale targeted targeting of endogenous genes with transcription repressors (CRISPRi) and activators (CRISPRa), and other applications of RNA-induced DNA targeting by Cas enzymes.

[0005] The CRISPR-Cas system is native to bacteria and archaea and provides adaptive immunity against viruses and plasmids. Three classes of the CRISPR-Cas system may be potentially adapted for research and therapeutic reagents. The type II CRISPR system has desirable features when utilizing a single CRISPR-associated (Cas) nuclease (specifically Cas9) in a complex with a suitable guide RNA (gRNA). In bacteria or archaea, the Cas9 guide RNA comprises two distinct RNA species. Target-specific CRISPR-activating RNA (crRNA) binds to a specific DNA sequence and guides the Cas9 / gRNA complex to target it. The crRNA has two functional domains: a target-specific 5' domain and a 3' domain that guides the binding of the crRNA to trans-activating crRNA (tracrRNA). TracrRNA is a longer universal RNA that binds to crRNA and mediates the binding of the gRNA complex to Cas9. Binding of tracrRNA induces a change in the Cas9 structure, shifting it from an inactive conformation to an active conformation. gRNA function can also be provided as an artificial single guide RNA (sgRNA) in which crRNA and tracrRNA are fused into a single species (see Jinek, M. et al., Science 337, pp. 816-821, 2012). The sgRNA format allows transcription of functional gRNA from a single transcription unit, which can be provided by a double-stranded DNA (dsDNA) cassette containing a transcription promoter and an sgRNA sequence. In mammalian systems, these RNAs have been introduced by transfection of RNA Pol III promoters (such as U6 or H1) that drive RNA transcription, viral vectors, and DNA cassettes containing single-stranded RNA after in vitro transcription (see Xu, T. et al., Appl Environ Microbiol, 2014, 80(5): pp. 1544-52).

[0006] In the CRISPR-Cas system, for example, using the system found in Streptococcus pyogenes (S.py. or Spy), the natural crRNA is approximately 42 nucleotides long and contains a 5' region of approximately 20 nucleotides long that is complementary to the target sequence (also referred to as the protospacer sequence or protospacer domain of the crRNA) and a 3' region of approximately 22 nucleotides long that is complementary to the tracrRNA sequence and mediates the binding of the crRNA to the tracrRNA. The crRNA:tracrRNA complex contains a functional gRNA that can lead to Cas9 cleavage of the complementary target DNA. The natural tracrRNA is approximately 85-90 nucleotides long and has a 5' region containing a region complementary to the crRNA. The remaining 3' region of the tracrRNA contains a secondary structure motif (referred to herein as the "tracrRNA 3' tail") that mediates the binding of the crRNA:tracrRNA complex to Cas9.

[0007] Jinek et al. conducted a comprehensive investigation of the physical domains of crRNA and tracrRNA required for proper functioning of the CRISPR-Cas system (Science, 2012, 337(6096): pp. 816-821). They devised cleaved crRNA:tracrRNA fragments that could still function in CRISPR-Cas, with the crRNA being 42 nucleotides in the wild type and the tracrRNA being cleaved into 75 nucleotides. They also developed embodiments in which crRNA and tracrRNA are linked to a linker loop to form a single guide RNA (sgRNA), which varies between 99 and 123 nucleotides in different embodiments.

[0008] At least three groups have elucidated the crystal structure of Streptococcus pyogenes Cas9 (SpyCas9). Jinek, M et al. found that this structure did not exhibit nuclease activity in complex with either guide RNA or target DNA. They conducted molecular modeling experiments to reveal predictive interactions between proteins in RNA and DNA complexes (Science, 2014, 343, 1215, DOI:10.1126 / science / 1247997).

[0009] Nishimasu, H et al. have shown the crystal structure of Spy Cas9 at 2.5 angstrom resolution in a complex of sgRNA and this target DNA (Cell, 2014, 156(5): pp. 935-949, incorporated herein by reference). This crystal structure identified two lobes for the Cas9 enzyme: a recognition lobe (REC) and a nucleature lobe (NUC). The sgRNA and target DNA heteroduplex (negatively charged) are located in a positively charged groove between the two lobes. The REC lobe, which does not show structural similarities to known proteins and is therefore a Cas9-specific functional domain, interacts with complementary crRNA and tracrRNA portions.

[0010] Another group, Briner et al. (Mol Cell, 2014, 56(2):333-339, the entire work is incorporated herein), identified and characterized six conserved modules within the natural crRNA:tracrRNA double helix and sgRNA. Anders et al. (Nature, 2014, 513(7519), pp. 569-5673) elucidated the structural basis for the recognition of protospacer-associated motif (PAM) sequences from sgRNA guides by Cas9.

[0011] The CRISPR-Cas endonuclease system is used in genome engineering as follows: a gRNA complex (either a crRNA:tracrRNA complex or sgRNA) binds to Cas9, activating Cas9 and inducing a conformational change that opens the DNA-binding cleft. The protospacer domain of the crRNA (or sgRNA) aligns with the complementary target DNA, Cas9 binds to the PAM sequence, and begins unwinding the target DNA. Subsequently, annealing of the protospacer domain to the target occurs, followed by cleavage of the target DNA. Cas9 contains two domains homologous to the endonucleases HNH and RuvC, respectively. The HNH domain cleaves the DNA strand complementary to the crRNA, while the RuvC-like domain cleaves the non-complementary strand. This results in double-strand breaks in genomic DNA. When repaired by non-homologous end joining (NHEJ), breaks are typically repaired in an imprecise manner, resulting in a DNA sequence shifted by one or more bases, leading to the breakdown of the native DNA sequence and, often, frameshift mutations if this event occurs within the coding exon of a protein-coding gene. Breaks can also be repaired by homology-directed recombination (HDR), which allows for the insertion of new genetic material based on exogenous DNA introduced into cells containing the Cas9 / gRNA complex, which is introduced into the break site created by the Cas9 break.

[0012] While the wild-type (WT) Cas9 protein cleaves most DNA targets with high efficiency, it exhibits levels of undesirable off-target editing sufficient to complicate research applications and raise significant concerns for medical use. In this context, off-target cleavage is defined as a DNA cleavage event occurring at a genomic DNA target site that is not perfectly complementary to the protospacer domain of the crRNA or sgRNA. Introducing cleavage events to untargeted sites via such off-target cleavage pathways is undesirable. Typically, cleavage is only desirable at genomic sites that are perfectly complementary to the gRNA. Several groups have published novel mutant Cas9 enzymes exhibiting reduced off-target cleavage activity (see Slaymaker et al., Science, 2016, 351, pp. 84-88; Kleinstiver et al., Nature, 2016, 529, pp. 490-495; Chen et al., Nature, 2017, http: / / dx.doi.org / 10.1038 / nature24268(2017)). The mutants described in these three publications were designed by selective mutations in specific amino acid residues in the Cas9 protein, which were identified as contact sites between the protein and the RNA guide and / or DNA substrate based on the crystal structure of the Cas9 protein. While understanding the mechanism of action is not required to carry out these inventions (i.e., to perform genome editing with improved specificity), it was initially thought that improved fidelity mutants act by reducing the relative affinity of the mutant Cas9 nuclease to substrate DNA compared to the WT enzyme, making it more likely that the mismatch between guide RNA and substrate DNA will be destabilized. More recently, it has been proposed that the mutation restricts the transfer of the Cas9 structure from an inactive conformation to an active conformation, and that this transfer does not occur relatively effectively in the presence of a mismatch between the RNA guide and the DNA target. Regardless of the mechanism, these mutant Cas9 enzymes exhibit reduced cleavage of target DNA with incomplete complementarity to the guide RNA, if necessary. However, this improved specificity comes at the cost of reduced on-target activity, which is undesirable.In all three examples of prior art disclosing Cas9 variants with improved specificity, genome editing using the CRISPR / Cas9 method was performed using plasmid or other expression-based methods, i.e., methods first described in 2013 (see Cong et al., Science, 2013, pp. 339, 819-823; Mali et al., Science, 2013, pp. 339, 823-826). However, it is now understood that plasmid systems introduce complexity to genome editing. For example, plasmids can be integrated into the host genome, thereby leading to other undesirable genomic changes, which can trigger innate immune responses and lead to cell death. For these and other reasons, plasmid systems are not ideal for research applications where precise editing is desired, nor are they practical for medical applications where such side effects are unacceptable. More recently, methods using ribonucleoprotein (RNP) complexes in which recombinant Cas9 protein is pre-complexed with synthetic gRNA have been shown to be preferable to using DNA-based expression constructs. The RNP method provides highly active genome editing with reduced side effects (see Cho et al., Genome Research, 2014, 24, pp. 132-141; Aida et al., Genome Biology, 2015, 16, pp. 87-98). Therefore, it is desirable to develop a high-fidelity genome editing method compatible with the RNP protocol. All previously cited published examples describing Cas9 mutants with improved specificity have utilized plasmid-based DNA expression cassettes to perform and study genome editing results. This method results in high levels of overexpression of the mutant Cas9 protein over long periods, increasing the apparent enzymatic activity of the mutant. The inventors hereby describe how these improved specificity Cas9 variants (eSpCas9(1.1) and Cas9-HF1) reduce enzyme activity when RNP is used to perform genome editing, resulting in significantly impaired cleavage of target DNA sites compared to cleavage by the WT Cas9 protein. In many cases, target sites that act with high efficiency using the WT Cas9 protein show no evidence of cleavage when using the mutant variants.Therefore, the publicly available mutant Cas9 proteins have limited utility for precise genome editing, especially when more medically relevant RNP methods are used. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Jinek, M. et al., Science 337, pp. 816-21, 2012 [Non-Patent Document 2] Xu, T. et al., Appl Environ Microbiol, 2014.80(5):1544~52 [Non-Patent Document 3] Science, 2012, 337(6096):816~21 [Non-Patent Document 4] Science, 2014, 343, 1215 pages, DOI:10.1126 / science / 1247997 [Non-Patent Document 5] Cell, 2014, 156(5):935-49 [Non-Patent Document 6] Mol Cell, 2014, 56(2):333~9 [Non-Patent Document 7] Nature, 2014, 513(7519), pp. 569-73 [Non-Patent Document 8] Slaymaker et al., Science, 2016, 351, pp. 84-88. [Non-Patent Document 9] Kleinstiver et al., Nature, 2016, 529, pp. 490-495. [Non-Patent Document 10] Chen et al., Nature, 2017, http: / / dx.doi.org / 10.1038 / nature24268(2017) [Non-Patent Document 11] Cong et al., Science, 2013, 339, pp. 819-823. [Non-Patent Document 12] Mali et al., Science, 2013, 339, pp. 823 - 826 [Non - Patent Document 13] Cho et al., Genome Research, 2014, 24, pp. 132 - 141 [Non - Patent Document 14] Aida et al., Genome Biology, 2015, 16, pp. 87 - 98 [Summary of the Invention] [Problems to be Solved by the Invention]

[0014] Therefore, there is still a need for a method to improve the specificity of Cas9 genome editing. In particular, when used in the RNP format, there is a need for a Cas9 variant that exhibits improved specificity while retaining a high enzyme activity similar to that of the WT Cas9 enzyme. [Means for Solving the Problems]

[0015] The present invention relates to a Cas9 mutant gene and polypeptide for use in a CRISPR system and methods of using them.

[0016] In a first aspect, an isolated mutant Cas9 protein is provided. The isolated mutant Cas9 protein is active in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR - associated protein endonuclease system (the "CRISPR / Cas endonuclease system"). The CRISPR / Cas endonuclease system exhibits reduced off - target editing activity and maintained on - target editing activity as compared to the wild - type CRISPR / Cas endonuclease system.

[0017] In a second embodiment, an isolated ribonucleoprotein (RNP) complex is provided. The RNP complex contains a mutant Cas9 protein and a gRNA complex. The isolated ribonucleoprotein complex is active as a CRISPR / Cas endonuclease system, and the resulting CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

[0018] In a third embodiment, isolated nucleic acids encoding a mutant Cas9 protein are provided. The mutant Cas9 protein is active in the CRISPR / Cas endonuclease system, which exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

[0019] In a fourth embodiment, a CRISPR / Cas endonuclease system is provided. The CRISPR / Cas endonuclease system comprises a mutant Cas9 protein and gRNA. Compared to the wild-type CRISPR / Cas endonuclease system, the CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity.

[0020] In a fifth embodiment, a method is provided for performing gene editing that reduces off-target editing activity and maintains on-target editing activity. This method includes contacting a candidate editing DNA target site locus with an active CRISPR / Cas endonuclease system having a mutant Cas9 protein complexed with a suitable gRNA (e.g., a crRNA:tracrRNA complex or sgRNA). The interaction may occur in any environment, e.g., in a living animal, living cell, or in vitro isolated DNA. [Brief explanation of the drawing]

[0021] [Figure 1]This is a schematic diagram of a bacterial gene screening process used to select mutant Cas9 sequences with high on-target cleavage activity and low off-target cleavage activity. [Figure 2] The figure shows exemplary single amino acid mutations in S. pyogenes Cas9 that reduce off-target editing when the Cas9 nuclease is delivered by plasmid (as described in Example 3). CRISPR / Cas9 editing experiments were performed using a series of mutant Cas9 plasmid expression cassettes (expressing WT Cas9 or the shown mutant Cas9) co-transfected with an Alt-R® crRNA:tracrRNA complex targeting the human EMX1 gene. Relative editing efficiency was measured 48 hours after transfection into HEK293 cells. Relative on-target editing efficiency is shown by the black bar on the left side of the Y-axis (on-target sequence 5'-GAGTCCGAGCAGAAGAAGAAGGG-3' (SEQ ID NO: 133)), and editing at known off-target sites is shown by the white bar on the right side of the Y-axis (off-target sequence 5'-GAGTTAGAGCAGAAGAAGAAAGG-3' (SEQ ID NO: 134), underlined nucleotides highlight PAM sites in the genomic DNA target, and bolded bases indicate base mismatches between on-target and off-target sites). Off-target sites were identified by Tsai et al. (Nature Biotechnology 33: pp. 187-197, 2015). Experimental conditions included 30 nM EMX1 crRNA:tracrRNA and 100 ng of Cas9 plasmid introduced by lipofection using 0.5 microliters of Transit X-2 per well. Error bars represent the standard error of the mean (n=3). [Figure 3]The figure shows exemplary single-amino acid mutations in S. pyogenes Cas9 that reduce off-target editing when the Cas9 nuclease is delivered by plasmid (as described in Example 3). CRISPR / Cas9 editing experiments were performed using a series of mutant Cas9 plasmid expression cassettes (expressing WT Cas9 or the shown mutant Cas9) co-transfected with an Alt-R® crRNA:tracrRNA complex targeting the human HEKSite4 locus. Relative editing efficiency was measured 48 hours after transfection into HEK293 cells. Relative on-target editing efficiency is shown by the black bar on the left side of the Y-axis (5'-GGCACTGCGGCTGGAGGTGGGGG-3' (SEQ ID NO: 135)), and editing at known off-target sites is shown by the white bar on the right side of the Y-axis (off-target sequence 5'-GGCACGACGGCTGGAGGTGGGGG-3' (SEQ ID NO: 136), underlined nucleotides highlight the PAM site in the genomic DNA target, and bolded bases indicate base mismatches between on-target and off-target sites). Off-target sites were identified by Tsai et al. (Nature Biotechnology 33: pp. 187-197, 2015). Experimental conditions included 30 nM EMX1 crRNA:tracrRNA and 100 ng of Cas9 plasmid introduced by lipofection using 0.5 microliters of Transit X-2 per well. Error bars represent the standard error of the mean (n=3). [Figure 4]This figure shows exemplary amino acid mutations in S. pyogenes Cas9 that reduce off-target editing by Cas9, resulting in the same or even reduced off-target editing when the mutant amino acid selected by bacterial screening is substituted with alanine. The Cas9 gRNA complex is delivered using an Alt-R® crRNA:tracrRNA complex targeting the EMX1 locus, as shown in Figure 2. Relative on-target editing efficiency is shown by the black bar on the left side of the Y axis (on-target sequence 5'-GAGTCCGAGCAGAAGAAGAAGGG-3' (SEQ ID NO: 133)), and editing at known off-target sites is shown by the white bar on the right side of the Y axis (off-target sequence 5'-GAGTTAGAGCAGAAGAAGAAAGG-3' (SEQ ID NO: 134), with underlined nucleotides highlighting PAM sites in the genomic DNA target and bolded bases indicating base mismatches at off-target sites relative to on-target sites). In each case, the bacterial-selected mutant and its alanine substitution counterpart are shown side by side. The experimental conditions and error analysis are the same as those shown in Figure 2. [Figure 5]This figure shows exemplary amino acid mutations in S. pyogenes Cas9 that reduce off-target editing by Cas9, resulting in the same or even reduced off-target editing when the mutant amino acid selected by bacterial screening is substituted with alanine. The Cas9 gRNA complex is delivered using an Alt-R® crRNA:tracrRNA complex targeting the HEKSite4 locus, as shown in Figure 3. Relative on-target editing efficiency is shown by the black bar on the left side of the Y axis (5'-GGCACTGCGGCTGGAGGTGGGGG-3' (SEQ ID NO: 135)), and editing at known off-target sites is shown by the white bar on the right side of the Y axis (off-target sequence 5'-GGCACGACGGCTGGAGGTGGGGG-3' (SEQ ID NO: 136), underlined nucleotides highlight PAM sites in genomic DNA targets, and bolded bases indicate base mismatches at off-target sites relative to on-target sites). In each case, the bacterial-selected mutant and its alanine substitution counterpart are shown side by side. The experimental conditions and error analysis are the same as those shown in Figure 3. [Figure 6]This figure shows an exemplary CRISPR / Cas9 editing experiment comparing different single and double amino acid mutations in the Cas9 protein, demonstrating relative on-target and off-target editing efficiencies. The Cas9 gRNA complex is delivered using an Alt-R® crRNA:tracrRNA complex targeting the HEKSite4 locus, as shown in Figure 3. Relative on-target editing efficiency is shown by the black bar on the left side of the Y-axis (5'-GGCACTGCGGCTGGAGGTGGGGG-3' (SEQ ID NO: 135)), and editing at known off-target sites is shown by the white bar on the right side of the Y-axis (off-target sequence 5'-GGCACGACGGCTGGAGGTGGGGG (SEQ ID NO: 136), with underlined nucleotides highlighting PAM sites in the genomic DNA target and bolded bases indicating base mismatches at the off-target site relative to the on-target site). Experimental conditions and error analysis are the same as those shown in Figures 2 and 3 and Figures 4 and 5. [Figure 7] This figure demonstrates that purified recombinant R691A Cas9 mutant protein exhibits superior on-target editing activity compared to known eSpCas9(1.1) or SpCas9-HF1 proteins when delivered as a ribonucleoprotein (RNP) complex. RNP complexes were formed using WT (black), eSpCas9(1.1) (white), SpCas9-HF1 (gray), or R691A (hatched) Cas9 proteins and Alt-R crRNA:tracrRNA gRNA complexes (crRNA SEQ ID NOs. 89-100) targeting different sites within the HRPT locus (1 μM). RNP delivery to cells was performed 48 hours prior to analysis in HEK293 cells with 10 nM RNP (Cas9:tracrRNA:crRNA (Alt-R crRNA) in a 1:1:1 ratio) along with 1.2 microliters of RNAiMAX lipofection. Error bars represent the standard error of the mean. [Figure 8]This figure demonstrates that purified recombinant R691A Cas9 mutant protein, when delivered as a ribonucleoprotein (RNP) complex, exhibits superior on-target editing activity compared to known eSpCas9(1.1) or SpCas9-HF1 proteins. RNP complexes were formed using WT (black), eSpCas9(1.1) (white), SpCas9-HF1 (gray), or R691A (hatched) Cas9 proteins and Alt-R crRNA:tracrRNA gRNA complexes (crRNA SEQ ID NOs. 101-112) targeting different sites within the CTLA4 locus (1 μM). Experimental details and error analysis are similar to those described in Figure 7. [Figure 9] This figure shows exemplary experimental results demonstrating that off-target editing activity is undetectable by T7EI analysis using R691A mutant Cas9 when delivered as RNP complexes to three independent, validated off-target sites for three different crRNAs. The RNP complexes were formed using WT (black), eSpCas9(1.1)(white), SpCas9-HF1 (gray), or R691A (hatched) Cas9 proteins and Alt-R crRNA:tracrRNA gRNA complexes targeting the human EMX1 (left) (SEQ ID NO: 113), HEKSite4 (center) (SEQ ID NO: 114), or VEGFA3 (right) (SEQ ID NO: 116) loci (1 μM). The on-target and off-target sites for the EMX1 and HEKSite4 loci are shown in Figures 2 and 3, respectively. On-target editing for known problematic off-target sites in the VEGFA3 locus (5'-GGTGAGTGAGTGTGTGCGTGTGG-3' (SEQ ID NO: 137)) and this guide is shown (5'-AGTGAGTGAGTGTGTGTGTGGGG-3' (SEQ ID NO: 138), underlined nucleotides indicate PAM sites, and the base differences between off-target and on-target sites are highlighted in bold font (Tsai et al., Nature Biotechnology 33: pp. 187-197, 2015). [Figure 10]This figure shows exemplary experimental results demonstrating that while the R691A mutant conventionally exhibits low-efficiency editing and thus maintains a high on-target editing activity guide site useful for distinguishing differences in on-target editing efficiency, combining this mutation with other amino acid changes identified in bacterial screening as double or triple mutants (i.e., combining it with selected mutations that reduce off-target editing activity by Cas9) also significantly reduces on-target editing activity. RNP complexes were formed with WT, R691A, N692A, T740A, S845A, S872A, R691A / T740A, R691A / S845A, R691A / S872A, R691A / N692A / T740A, R691A / N692A / S845A, or R691A / N692A / S872A Cas9 proteins (1 μM). RNP complexes (10 nM) were delivered into HEK293 cells by reverse transfection using RNAiMAX, and DNA was extracted after 48 hours. Error bars represent the standard error of the mean. [Figure 11]This figure shows exemplary experimental results demonstrating that while the R691A mutant maintains high on-target editing activity at the problematic on-target site, combining this mutation with other amino acid changes identified in bacterial screening as a double or triple mutant (i.e., combining it with selected mutations that reduce off-target editing activity by Cas9) significantly reduces on-target editing activity. RNP complexes were formed with WT, R691A, N692A, T740A, S845A, S872A, R691A / T740A, R691A / S845A, R691A / S872A, R691A / N692A / T740A, R691A / N692A / S845A, or R691A / N692A / S872A Cas9 proteins (1 μM). RNP complexes (10 nM) were delivered into HEK293 cells by reverse transfection using RNAiMAX, and DNA was extracted after 48 hours. Error bars represent the standard error of the mean. On-target and off-target sites for the HEKSite4 locus are shown in Figure 3. The details of the experiment and the error analysis are the same as those shown in Figure 9. [Figure 12]This figure shows exemplary experimental results demonstrating the effect of introducing any possible amino acid substitution mutation at the R691 position on editing at the HPRT 38509 site, where many substitutions maintain on-target editing activity, while others impair editing efficiency. The Cas9 protein was delivered by plasmid, and the CRISPR / Cas9 editing experiments used plasmid-mediated Cas9 (expressing WT "R691" Cas9 (SEQ ID NO: 5) or the shown mutant Cas9 (SEQ ID NOs: 7, 71-88)) co-transfected with an Alt-R® crRNA:tracrRNA complex targeting the HPRT38509 locus (SEQ ID NO: 92). Experimental conditions included 30 nM HPRT 38509 crRNA introduced by lipofection using 0.5 microliters of Transit X-2 per well and 100 ng of Cas9 plasmid, with cells incubated 48 hours prior to analysis. Error bars represent the standard error of the mean. [Figure 13] This figure shows exemplary experimental results demonstrating the effect of introducing any possible amino acid substitution mutation at the R691 position on editing at the HEKSite4 site, where the majority of mutations dramatically reduce off-target editing while also maintaining on-target activity. The Cas9 protein was delivered by plasmid, and the CRISPR / Cas9 editing experiments used plasmid-mediated Cas9 (expressing WT "R691" Cas9 (SEQ ID NO: 5) or the shown mutant Cas9 (SEQ ID NOs: 7, 71-88)) and co-transfected with an Alt-R® crRNA:tracrRNA complex targeting the HEKSite4 locus (SEQ ID NO: 114). Experimental conditions included 30 nM HEKSite4 crRNA introduced by lipofection and 100 ng of Cas9 plasmid in 0.5 microliters of Transit X-2 per well, with cells incubated 48 hours prior to analysis. Error bars represent the standard error of the mean. On-target and off-target sites for the HEKSite4 locus are shown in Figure 3. The details of the experiment and the error analysis are the same as those shown in Figure 9. [Figure 14]This figure shows exemplary experimental results demonstrating that selected Cas9 mutant proteins with different amino acid substitutions at the R691 position maintain on-target editing activity at different guide sites within the human HPRT gene. RNP complexes were formed (1 μM) with WT (R691), R691A, R691D, R691G, R691H, R691Y, or R691W Cas9 proteins (sequences 5, 7, 73, 77, 78, 87, and 86, respectively) using Alt-R® crRNA:tracrRNA gRNA complexes targeting either the HRPT 38509 or HPRT 38087 locus (sequences 92 and 94). RNP complexes (10 nM) were delivered into HEK293 cells by reverse transfection using RNAiMAX, and DNA was extracted after 48 hours. Error bars represent the standard error of the mean. Experimental details and error analysis are similar to those described in Figure 9. [Figure 15] This figure shows exemplary experimental results demonstrating that selected Cas9 mutant proteins with different amino acid substitutions at the R691 position maintain on-target editing activity and reduce off-target editing activity with multiple guides. RNP complexes were formed with WT (R691), R691A, R691D, R691G, R691H, R691Y, or R691W Cas9 proteins (sequences 5, 7, 73, 77, 78, 87, and 86, respectively) using Alt-R® crRNA:tracrRNA gRNA complexes targeting the HEKSite4 (sequence number 114) or EMX1 (sequence number 113) loci (1 μM). RNP complexes (10 nM) were delivered into HEK293 cells by reverse transfection using RNAiMAX, and DNA was extracted after 48 hours. On-target and off-target sites for the EMX1 and HEKSite4 loci are shown in Figures 2 and 3, respectively. Experimental details and error analysis are similar to those shown in Figure 9. [Figure 16]This figure shows exemplary reductions in off-target editing activity in unbiased and genome-wide settings using the previously described GUIDE-Seq procedure (Tsai et al., Nature Biotechnology 33: pp. 187-197, 2015) in living cells. RNP complexes were formed with WT (SEQ ID NO: 6) or R691A (SEQ ID NO: 8) Cas9 protein using Alt-R® crRNA:tracrRNA gRNA complexes targeting either the EMX1 (SEQ ID NO: 113), VEGFA3 (SEQ ID NO: 116), or AR (SEQ ID NO: 115) gene guide sites (1 μM). RNP complexes (4 μM) were delivered into HEK293 cells along with 0.5 μM dsDNA GUIDE-Seq tags (SEQ ID NOs: 139 and 140) by electroporation using a Lonza Nucleofector, and the DNA was extracted after 48 hours. NGS library construction, sequencing, and data analysis were carried out as previously described (Tsai et al., Nature Biotechnology 33: pp. 187-197, 2015). [Modes for carrying out the invention]

[0022] The methods and compositions of the present invention described herein provide mutant SpyCas9 nucleic acids and polypeptides for use in CRISPR-Cas systems. The present invention describes novel Cas9 variants that maintain high on-target editing activity compared to wild-type proteins, even when delivered as RNP complexes, while reducing off-target editing activity to low levels. These and other advantages of the present invention, as well as further features of the present invention, will become apparent from the description of the invention provided herein.

[0023] The term "wild-type Cas9 protein" ("WT-Cas9" or "WT-Cas9 protein") encompasses proteins that have the same amino acid sequence as naturally occurring Streptococcus pyogenes Cas9 (e.g., SEQ ID NO: 5) and possess biochemical and biological activity when combined with appropriate guide RNA (e.g., sgRNA or dual crRNA:tracrRNA composition) to form an active CRISPR-Cas endonuclease system.

[0024] The term "wild-type CRISPR / Cas endonuclease system" refers to a CRISPR / Cas endonuclease system that includes the wild-type Cas9 protein and appropriate gRNA.

[0025] The phrase "an active CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to a wild-type CRISPR / Cas endonuclease system" refers to the activity of a CRISPR / Cas endonuclease system containing a mutant Cas9 protein that typically exhibits a greater reduction in off-target editing activity than the corresponding reduction in on-target editing activity compared to a wild-type CRISPR / Cas endonuclease system containing a wild-type Cas9 protein, when both CRISPR / Cas endonuclease systems contain the same gRNA for a given target sequence. The preferred off-target and on-target editing activity of a CRISPR / Cas endonuclease system depends on the target gRNA and target sequence, and such preferred off-target and on-target editing activity of a CRISPR / Cas endonuclease system with a mutant Cas9 protein is illustrated in the examples.

[0026] The term "mutant Cas9 protein" encompasses protein forms that have a different amino acid sequence from wild-type Streptococcus pyogenes Cas9 and possess biochemical and biological activity when combined with appropriate guide RNA (e.g., sgRNA or dual crRNA:tracrRNA composition) to form an active CRISPR-Cas endonuclease system. This includes orthologs and Cas9 variants that have a different amino acid sequence from wild-type Streptococcus pyogenes Cas9.

[0027] The mutant Cas9 protein amino acid sequences referred to herein include those expressed as full-length amino acid sequences, as presented in this disclosure and the sequence listing. However, for brevity, abbreviated mutant Cas9 protein amino acid coding is provided herein, and the position and identity of a given substitution mutation are provided relative to the amino acid position and identity of the wild-type Cas9 protein amino acid sequence (e.g., SEQ ID NO: 5). For example, a single substitution mutation introduced at R691 in the wild-type Cas9 protein amino acid sequence refers to a substitution mutation that replaces arginine at residue 691 in the wild-type Cas9 protein amino acid sequence. For example, a specific single substitution mutation R691A refers to a mutant Cas9 protein amino acid sequence containing alanine instead of arginine at residue 691 of the wild-type Cas9 protein amino acid sequence (see, for example, SEQ ID NO: 7).

[0028] The mutant Cas9 protein of the present invention is active in the CRISPR / Cas endonuclease system, and the resulting CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system. Where used herein, “mutant Cas9 protein” excludes the mutant Cas9 proteins disclosed in Slaymaker et al., Science, 2016, 351, pp. 84-88; Kleinstiver et al., Nature, 2016, pp. 529, pp. 490-495; and Chen et al., Nature, 2017, http: / / dx.doi.org / 10.1038 / nature24268(2017)) to the extent that these mutant Cas9 proteins exhibit reduced off-target editing activity and maintained on-target editing activity compared to wild-type CRISPR / Cas endonuclease systems, and to the extent that the mutant Cas9 proteins disclosed herein are considered statutory “prior art” with respect to this application and U.S. Provisional Patent Application No. 62 / 405,601.For example, as used herein and subject to the above conditions, “mutant Cas9 protein” means, in particular, K775A, R780A, K810A, R832A, K848A, K855A, K862A, K961A, K968A, K974A, R976A, H982A, K1003A, K1014A, K1047A, K1059A, R1060A, H1241A, K1289A, K1296A, H1297A, K1300A, H1311A, K1325A, eSpCas9(1.0)(K810A / K1003A / R1060A), eSpCas9(1.1)(K8 Mutant Cas9 proteins selected from the group consisting of 48A / K1003A / R1060A), SpCas9-HF1 (N497A / R661A / Q695A / Q926A) and Hypa-Cas9 (N692A / M694A / Q695A / H698A; "Cluster 1"), Cluster 2 (G582A / V583A / E584A / D585A / N588A), Cluster 3 (T657A / R661A / G658A / W659A), Cluster 4 (N497A / F491A / M495A / T496A), and Cluster 5 (K918A / V922A / R925A) will be excluded.

[0029] The term "polypeptide" refers to any linear or branched peptide containing more than one amino acid. Polypeptides include proteins, fragments, or fusions thereof, provided that such proteins, fragments, or fusions retain useful biochemical or biological activity.

[0030] Fusion proteins typically contain additional amino acid information that is not native to the original protein, and this additional amino acid information is covalently bound to the protein. Such additional amino acid information may include tags that enable the purification or identification of the fusion protein. Such additional amino acid information may include peptides that enable the fusion protein to be transported into a cell and / or to a specific location within the cell. Examples of tags for these purposes include: AviTag (GLNDIFEAQKIEWHE), a peptide that enables biotinylation by the enzyme BirA so that the protein can be isolated by streptavidin; calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL), a peptide to which the protein calmodulin is bound; polyglutamate tag (EEEEEE), a peptide that effectively binds to anion exchange resins such as Mono-Q; E tag (GAPVPYPDPLEPR), a peptide recognized by antibodies; FLAG tag (DYKDDDDK), a peptide recognized by antibodies; HA tag (YPYDVPDYA), a hemagglutinin-derived peptide recognized by antibodies; and 5-10 histidines typically bound to nickel or cobalt chelates. A His tag (HHHHHH); a Myc tag (EQKLISEEDL), a peptide derived from c-myc recognized by an antibody; a NE tag (TKENPRSNQEESYDDNES), a novel 18-amino acid synthetic peptide recognized by a monoclonal IgG1 antibody, useful for a wide range of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and recombinant protein affinity purification; an S tag (KETAAAKFERQHMDS), a peptide derived from ribonuclease A; an SBP tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), a peptide bound to streptavidin; Softag1 (SLAELLNAGLGGS), intended for mammalian expression; Softag3 (TQDPSRVG), intended for prokaryotic expression.Strep tag (Strep tag II: WSHPQFEK), a peptide bound to modified streptavidin called streptavidin or streptactin; FlAsH and ReAsH, peptides recognized by antibodies; TC tag (GKPIPNPLLGLDST), a tetracysteine ​​tag recognized by the biarsenic compound (CCPGCC) V5 tag; VSV tag (YTDIEMNRLGK); Xpress tag (DLYDDDDK), peptides recognized by antibodies; Isopep tag (TDKDMTITFTNKKDAE), a peptide covalently bound to pyrin-C protein; SpyTag (AHIVMVDA), a peptide covalently bound to SpyCatcher protein. YKPTK; SnoopTag (KLGDIEFIKVNK), a peptide covalently bound to the SnoopCatcher protein; BCCP (Biotin Carboxylate Carrier Protein), a protein domain biotinylated by BirA to enable recognition by streptavidin; Glutathione-S-Transferase Tag, a protein bound to immobilized glutathione; Green Fluorescent Protein Tag, a naturally fluorescent protein to which antibodies can bind; HaloTag, a mutant bacterial haloalkane dehalogenase covalently bound to a reactive haloalkane substrate to enable attachment to a wide variety of substrates; Maltose-Binding Protein Tag, a protein bound to amylose agarose; Nus Tag; Thioredoxin Tag;Furthermore, Fc tags derived from immunoglobulin Fc domains, which enable dimerization and solubilization and can be used for purification on Protein A Sepharose. Nuclear localization signals (NLS), such as those obtained from SV40, allow the protein to be transported to the nucleus as soon as it enters the cell. Assuming that the native Cas9 protein is of bacterial origin and therefore does not naturally contain NLS motifs, the addition of one or more NLS motifs to a recombinant Cas9 protein is expected to exhibit improved genome editing activity when used in eukaryotic cells where the target genomic DNA substrate resides in the nucleus. Those skilled in the art will understand how to construct and use these various fusion tag technologies and fusion proteins containing them.

[0031] The term “isolated nucleic acids” includes DNA, RNA, cDNA, and vectors encoding them, and does not include other biological materials from which they may originate or be related, such as cellular components. Typically, isolated nucleic acids are purified from other biological materials from which they may originate or be related, such as cellular components.

[0032] The term "isolated wild-type Cas9 nucleic acid" refers to an isolated nucleic acid that encodes the wild-type Cas9 protein. Examples of isolated wild-type Cas9 nucleic acids include SEQ ID NOs: 1 and 2.

[0033] The term "isolated mutant Cas9 nucleic acid" refers to an isolated nucleic acid that encodes a mutant Cas9 protein. Examples of isolated mutant Cas9 nucleic acids include SEQ ID NOs: 3 and 4.

[0034] The term "length modified" refers, when used to modify RNA, to a shortened or cleaved form of a reference RNA lacking a nucleotide sequence, or an extended form of a reference RNA containing an additional nucleotide sequence.

[0035] The term "chemically modified" refers to a form of reference RNA containing a chemically modified nucleotide or non-nucleotide chemical group covalently linked to the RNA, when this term modifies RNA. Chemically modified RNA, as described herein, generally refers to synthetic RNA prepared using oligonucleotide synthesis procedures in which the modified nucleotide is incorporated during the synthesis of the RNA oligonucleotide. However, chemically modified RNA also includes synthetic RNA oligonucleotides that have been modified after synthesis with appropriate modifying agents.

[0036] A competent CRISPR-Cas endonuclease system comprises a ribonucleoprotein (RNP) complex formed with an isolated guide RNA selected from either an isolated Cas9 protein and a combination of dual crRNA:tracrRNA or a chimeric single molecule sgRNA. In some embodiments, isolated length-modified and / or chemically modified forms of crRNA and tracrRNA are combined with purified Cas9 protein, isolated mRNA encoding the Cas9 protein, or the gene encoding the Cas9 protein in an expression vector. In certain assays, isolated length-modified and / or chemically modified forms of crRNA and tracrRNA can be introduced into cell lines stably expressing the Cas9 protein from an endogenous expression cassette encoding the Cas9 gene. In other assays, a mixture of length-modified and / or chemically modified forms of crRNA and tracrRNA combined with either mutant Cas9 mRNA or mutant Cas9 protein can be introduced into cells.

[0037] The applicant has previously presented novel crRNA and tracrRNA oligonucleotide compositions exhibiting potent activity in the CRISPR-Cas endonuclease system, which consists of short repeat palindromic sequences clustered at regular intervals. The oligonucleotide compositions include length-modified forms of crRNA and tracrRNA, as well as chemically modified forms of crRNA and tracrRNA. Length-modified forms of crRNA and tracrRNA enable the preparation of active forms of these RNAs using conventionally available, cost-effective, and efficient oligonucleotide synthesis protocols. Chemically modified forms of crRNA and tracrRNA provide activators that can be tuned to certain specific intrinsic properties, such as improved stability in cellular and in vivo environments, or a reduced risk of inducing innate immune responses in mammalian cells. Length-modified forms of crRNA and tracrRNA may also include modifications, thereby enabling their use in a wide range of compositions active in the CRISPR-Cas endonuclease system environment. These oligonucleotide compositions and their properties in the CRISPR-Cas endonuclease system may be used in conjunction with the mutant Cas9 nucleic acids and proteins disclosed herein. These oligonucleotide compositions and their properties in the CRISPR-Cas endonuclease system are disclosed in U.S. Patent Application No. 14 / 975,709, filed on 18 December 2015, entitled "CRISPR-BASED COMPOSITIONS AND METHODS OF USE," published on 23 June 2016 as U.S. Patent Publication No. US2016-0177304 A1 by Collingwood et al. (Applicant: Integrated DNA Technologies, Inc. (Skokie, IL (US))), and currently issued as U.S. Patent No. __________, which are incorporated herein by reference in their entirety.

[0038] Mutant Cas9 protein with reduced off-target gene editing activity In a first embodiment, an isolated mutant Cas9 protein is provided. The isolated mutant Cas9 protein is active in a short repeat palindromic sequence (CRISPR) / CRISPR-related protein endonuclease system ("CRISPR / Cas endonuclease system") clustered at regular intervals. The resulting CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

[0039] Preferred single-mutant Cas9 proteins include substitutional mutations in WT-Cas9 introduced at one of the following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872, and R925. Exemplary single-mutant Cas9 proteins include the following specific mutations introduced into WT-Cas9: R494C, R494A, N522K, N522A, N588D, N588A, N612A, T657A, S663A, R691S, R691A, N692D, N692A, S730G, S730A, T740A, R765G, R765A, T770K, T770A, N776A, R778A, R783A, S793A, N803D, N803A, S845A, N854K, N854A, S872A, R925C, and R925A. Exemplary single-mutant Cas9 proteins include at least one member selected from the group consisting of SEQ ID NOs: 7-38. Further substitution mutations may be included in the amino acid background of the single mutant Cas9 protein amino acid sequence, provided that the resulting mutant Cas9 protein is active as a CRISPR / Cas endonuclease system, and that the resulting CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

[0040] Preferred double mutant Cas9 proteins include mutations in WT-Cas9 introduced at two of the following locations: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872, and R925. The most desirable double mutant Cas9 proteins include mutations in WT-Cas9 introduced at the following positions: R691 / N692, R691 / R494, R691 / N522, R691 / N588, R691 / N612, R691 / S663, R691 / T730, R691 / T740, R691 / R765, R691 / T770, N692 / T740, R691 / S845, N692 / S845, R691 / S872, and N692 / S872. The exemplary double mutant Cas9 protein contains two distinct specific mutations introduced into WT-Cas9 selected from the following amino acid mutations: R494C or R494A; N522K or N522A; N588D or N588A; N612A; T657A; S663A; R691S or R691A; N692D or N692A; S730G or S730A; T740A; R765G or R765A; T770K or T770A; N776A; R778A; R783A; S793A; N803D or N803A; S845A; N854K or N854A; S872A; and R925C or R925A. An exemplary double mutant Cas9 protein contains at least one member selected from the group consisting of SEQ ID NOs: 39–88. Further substitutional mutations may be included in the amino acid background of the double mutant Cas9 protein amino acid sequence, provided that the resulting mutant Cas9 protein is active as a CRISPR / Cas endonuclease system, and that the resulting CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

[0041] In a second embodiment, an isolated ribonucleoprotein complex is provided. The RNP comprises a mutant Cas9 protein and a gRNA complex. In the first aspect, the gRNA comprises crRNA and tracrRNA in a stoichiometric (1:1) ratio. In the second aspect, the crRNA comprises Alt-R® crRNA (Integrated DNA Technologies, Inc. (Skokie, IL(US))) for a specific editing target site for a given locus, and the tracrRNA comprises Alt-R® tracrRNA (Integrated DNA Technologies, Inc. (Skokie, IL(US))). In another aspect, the gRNA comprises sgRNA. Preferred mutant Cas9 proteins include those described above.

[0042] In a third embodiment, isolated nucleic acids encoding mutant Cas9 proteins are provided. Preferred isolated nucleic acids encode the mutant Cas9 proteins described above. Exemplary isolated nucleic acids encoding mutant Cas9 proteins can be readily produced from nucleic acids encoding wild-type Cas9 proteins using recombinant DNA procedures or chemical synthesis methods. Preferred nucleic acids for this purpose include nucleic acids optimized for the expression of Cas9 proteins in bacterial (e.g., E. coli) or mammalian (e.g., human) cells. Exemplary codon-optimized nucleic acids for expressing WT-Cas9 (SEQ ID NO: 5) in E. coli and human cells include SEQ ID NOs: 1 and 2, respectively. Exemplary codon-optimized nucleic acids for expressing mutant Cas9 proteins (e.g., R691A mutant Cas9 protein; SEQ ID NO: 7) in E. coli and human cells include SEQ ID NOs: 3 and 4, respectively. Furthermore, the present invention intends to provide a fusion protein of WT-Cas9 and mutant Cas9, in which the coding sequences of WT-Cas9 and mutant Cas9 are fused with an amino acid sequence that encodes nuclear localization ("NLS") of the fusion protein in eukaryotic cells or an amino acid sequence that facilitates the purification of the protein. Exemplary fusion proteins containing either the WT-Cas9 amino acid sequence or the mutant Cas9 amino acid sequence (e.g., the R691A mutant Cas9 protein) include SEQ ID NOs. 6 and 8, respectively.

[0043] In the first point, the isolated nucleic acid contains mRNA encoding one of the aforementioned mutant Cas9 proteins. In the second point, the isolated nucleic acid contains DNA encoding a gene for one of the aforementioned mutant Cas9 proteins. Preferred DNA contains a vector encoding a gene encoding a mutant Cas9 protein. Such delivery methods include plasmids and various viral delivery vectors, as are well known to those skilled in the art. The mutant Cas9 protein can also be stably transformed into cells using a suitable expression vector to produce cell lines that constitutively or inductively express the mutant Cas9. The aforementioned method can also be applied to embryos to produce offspring animals that constitutively or inductively express the mutant Cas9.

[0044] In a fourth embodiment, a CRISPR / Cas endonuclease system is provided. The CRISPR / Cas endonuclease system comprises a mutant Cas9 protein. Preferred mutant Cas9 proteins include those described above. In a first aspect, the CRISPR / Cas endonuclease system is encoded by a DNA expression vector. In one embodiment, the DNA expression vector is a plasmid-borne vector. In a second embodiment, the DNA expression vector is selected from bacterial expression vectors and eukaryotic cell expression vectors. In a third aspect, the CRISPR / Cas endonuclease system comprises a ribonucleoprotein complex containing a mutant Cas9 protein and a gRNA complex. In a first aspect, the gRNAs include crRNA and tracrRNA in a stoichiometric (1:1) ratio. In the second respect, crRNA includes Alt-R® crRNA (Integrated DNA Technologies, Inc. (Skokie, IL(US))) for a specific editing target site for a given gene locus, and tracrRNA includes Alt-R® tracrRNA (Integrated DNA Technologies, Inc. (Skokie, IL(US))). In another respect, gRNA includes sgRNA.

[0045] In a fifth embodiment, a method is provided for performing gene editing that reduces off-target editing activity and / or increases on-target editing activity. This method comprises the step of contacting a candidate editing target site locus with an active CRISPR / Cas endonuclease system having a mutant Cas9 protein. In the first aspect, this method comprises a single mutant Cas9 protein having a mutation in WT-Cas9 introduced at one of the following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925. Exemplary single-mutant Cas9 proteins include the following specific mutations introduced into WT-Cas9: R494C, R494A, N522K, N522A, N588D, N588A, N612A, T657A, S663A, R691S, R691S, R691A, N692D, N692A, S730G, S730A, T740A, R765G, R765A, T770K, T770A, N776A, R778A, R783A, S793A, N803D, N803A, S845A, N854K, N854A, S872A, R925C, and R925A. Exemplary single-mutant Cas9 proteins include at least one member selected from the group consisting of SEQ ID NOs: 7-38. Further substitution mutations may be included in the amino acid background of the single mutant Cas9 protein amino acid sequence, provided that the resulting mutant Cas9 protein is active as a CRISPR / Cas endonuclease system in this method, and that the resulting CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

[0046] In other words, this method involves a double mutant Cas9 protein having mutations in WT-Cas9 introduced at two of the following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872, and R925. The most desirable double mutant Cas9 proteins include mutations in WT-Cas9 introduced at the following positions: R691 / N692, R691 / R494, R691 / N522, R691 / N588, R691 / N612, R691 / S663, R691 / T730, R691 / T740, R691 / R765, R691 / T770, N692 / T740, R691 / S845, N692 / S845, R691 / S872, and N692 / S872. The exemplary double mutant Cas9 protein contains two distinct specific mutations introduced into WT-Cas9 selected from the following amino acid mutations: R494C or R494A; N522K or N522A; N588D or N588A; N612A; T657A; S663A; R691S or R691A; N692D or N692A; S730G or S730A; T740A; R765G or R765A; T770K or T770A; N776A; R778A; R783A; S793A; N803D or N803A; S845A; N854K or N854A; S872A; and R925C or R925A. An exemplary double mutant Cas9 protein contains at least one member selected from the group consisting of SEQ ID NOs: 39–88. Further substitutional mutations may be included in the amino acid background of the double mutant Cas9 protein amino acid sequence, provided that the resulting mutant Cas9 protein is active as a CRISPR / Cas endonuclease system in this method, and that the resulting CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

[0047] The applications of Cas9-based methods are numerous and diverse. These include, but are not limited to, plant gene editing, yeast gene editing, mammalian gene editing, cell editing in organs of living animals, embryo editing, rapid generation of knockout / knock-in animal lines, generation of animal models of disease states, correction of disease states, insertion of reporter genes, and whole-genome functional screening. [Examples]

[0048] [Example 1] DNA and amino acid sequences of wild-type and mutant Cas9 proteins. The following list shows different wild-type (WT) and mutant Cas9 nucleases described in the present invention. It will be understood by those skilled in the art that many different DNA sequences can encode / express the same amino acid (AA) sequence, since often more than one codon can encode the same amino acid. The DNA sequences shown below are given only as examples, and other DNA sequences encoding the same protein (e.g., the same amino acid sequence) are intended. It will be further understood that further features, elements, or tags, such as NLS domains, may be added to the sequences. Examples are shown for WT Cas9 and mutant R691A Cas9, showing the amino acid and DNA sequences for these proteins as Cas9 alone and Cas9 fused with both C-terminal and N-terminal SV40NLS domains and the HIS tag. For other Cas9 variants, only the amino acid sequences are provided, but similar additions of NLS and His tag domains may be added to facilitate use in producing recombinant proteins for use in mammalian cells. Mutations different from the WT sequence are clearly indicated using underlined bold font.

[0049] Sequence ID 1 WT SpyCas9 DNA sequence codon-optimized for expression in E. coli.

[0050] [ka] JPEG2026065115000003.jpg7275

[0051] Sequence ID 2 A codon-optimized WT SpyCas9 DNA sequence for expression in H. sapiens.

[0052] [ka] JPEG2026065115000005.jpg8375

[0053] Sequence ID 3 R691A mutant SpyCas9 DNA sequence codon-optimized for expression in E. coli.

[0054] [ka] JPEG2026065115000007.jpg11272

[0055] Sequence ID 4 R691A mutant SpyCas9 DNA sequence codon-optimized for expression in H. sapiens.

[0056] [ka] JPEG2026065115000009.jpg1375

[0057] Sequence ID 5 WT SpyCas9 AA sequence.

[0058] [ka]

[0059] Sequence ID 6 WT SpyCas9 AA sequence with added NLS domain and HIS-Tag purification domain.

[0060] [ka]

[0061] Sequence ID 7 R691A mutant SpyCas9 AA sequence.

[0062] [ka]

[0063] Sequence ID 8 The R691A mutant SpyCas9 AA sequence with added NLS domain and HIS-Tag purification domain.

[0064] [ka]

[0065] Sequence ID 9 R494C mutant SpyCas9 AA sequence.

[0066] [ka]

[0067] Sequence ID 10 R494A mutant SpyCas9 AA sequence.

[0068] [ka]

[0069] Sequence ID 11 N522K mutant SpyCas9 AA sequence.

[0070] [ka]

[0071] Sequence ID 12 N522A mutant SpyCas9 AA sequence.

[0072] [ka]

[0073] Sequence ID 13 N588D mutant SpyCas9 AA sequence.

[0074] [ka]

[0075] Sequence ID 14 N588A mutant SpyCas9 AA sequence.

[0076] [ka]

[0077] Sequence ID 15 N612A mutant SpyCas9 AA sequence.

[0078] [ka]

[0079] Sequence ID 16 T657A mutant SpyCas9 AA sequence.

[0080] [ka]

[0081] Sequence ID 17 S663A mutant SpyCas9 AA sequence.

[0082] [ka]

[0083] Sequence ID 18 N692D mutant SpyCas9 AA sequence.

[0084] [ka]

[0085] Sequence ID 19 N692A mutant SpyCas9 AA sequence.

[0086] [ka]

[0087] Sequence ID 20 S730G mutant SpyCas9 AA sequence.

[0088] [ka]

[0089] Sequence ID 21 S730A mutant SpyCas9 AA sequence.

[0090] [ka]

[0091] Sequence ID 22 T740A mutant SpyCas9 AA sequence.

[0092] [ka]

[0093] Sequence ID 23 R765G mutant SpyCas9 AA sequence.

[0094] [ka]

[0095] Sequence ID 24 R765A mutant SpyCas9 AA sequence.

[0096] [ka]

[0097] Sequence ID 25 T770K mutant SpyCas9 AA sequence.

[0098] [ka] Sequence ID 26 T770A mutant SpyCas9 AA sequence.

[0099] [ka]

[0100] Sequence ID 27 N776A mutant SpyCas9 AA sequence.

[0101] [ka]

[0102] Sequence ID 28 R778A mutant SpyCas9 AA sequence.

[0103] [ka]

[0104] Sequence ID 29 R783A mutant SpyCas9 AA sequence.

[0105] [ka]

[0106] Sequence ID 30 S793A mutant SpyCas9 AA sequence.

[0107] [ka]

[0108] Sequence ID 31 N803D mutant SpyCas9 AA sequence.

[0109] [ka]

[0110] Sequence ID 32 N803A mutant SpyCas9 AA sequence.

[0111] [ka]

[0112] Sequence ID 33 S845A mutant SpyCas9 AA sequence.

[0113] [ka]

[0114] Sequence ID 34 N854K mutant SpyCas9 AA sequence.

[0115] [ka]

[0116] Sequence ID 35 N854A mutant SpyCas9 AA sequence.

[0117] [ka]

[0118] Sequence ID 36 S872A mutant SpyCas9 AA sequence.

[0119] [ka]

[0120] Sequence ID 37 R925C mutant SpyCas9 AA sequence.

[0121] [ka]

[0122] Sequence ID 38 R925A mutant SpyCas9 AA sequence.

[0123] [ka]

[0124] Sequence ID 39 R691A / N692A mutant SpyCas9 AA sequence.

[0125] [ka]

[0126] Sequence ID 40 R691A / R494C mutant SpyCas9 AA sequence.

[0127] [ka]

[0128] Sequence ID 41 N692A / R494C mutant SpyCas9 AA sequence.

[0129] [ka]

[0130] Sequence ID 42 R691A / N522K mutant SpyCas9 AA sequence.

[0131] [ka]

[0132] Sequence ID 43 N692A / N522K mutant SpyCas9 AA sequence.

[0133] [ka]

[0134] Sequence ID 44 R691A / N588D mutant SpyCas9 AA sequence.

[0135] [ka]

[0136] Sequence ID 45 N692A / N588D mutant SpyCas9 AA sequence.

[0137] [ka]

[0138] Sequence ID 46 R691A / N612A mutant SpyCas9 AA sequence.

[0139] [ka]

[0140] Sequence ID 47 N692A / N612A mutant SpyCas9 AA sequence.

[0141] [ka]

[0142] Sequence ID 48 R691A / S663A mutant SpyCas9 AA sequence.

[0143] [ka]

[0144] Sequence ID 49 N692A / S663A mutant SpyCas9 AA sequence.

[0145] [ka]

[0146] Sequence ID 50 R691A / S730G mutant SpyCas9 AA sequence.

[0147] [ka]

[0148] Sequence ID 51 N692A / S730G mutant SpyCas9 AA sequence.

[0149] [ka]

[0150] Sequence ID 52 R691A / T740A mutant SpyCas9 AA sequence.

[0151] [ka]

[0152] Sequence ID 53 N692A / T740A mutant SpyCas9 AA sequence.

[0153] [ka]

[0154] Sequence ID 54 R691A / R765G mutant SpyCas9 AA sequence.

[0155] [ka]

[0156] Sequence ID 55 N692A / R765G mutant SpyCas9 AA sequence.

[0157] [ka]

[0158] Sequence ID 56 R691A / T770K mutant SpyCas9 AA sequence.

[0159] [ka]

[0160] Sequence ID 57 N962A / T770K mutant SpyCas9 AA sequence.

[0161] [ka]

[0162] Array number 58 R691A / S793A mutant SpyCas9 AA sequence.

[0163]

Chemical Structure

[0164] Array number 59 N692A / S793A mutant SpyCas9 AA sequence.

[0165]

Chemical Structure

[0166] Array number 60 R691A / N803D mutant SpyCas9 AA sequence.

[0167]

Chemical Structure

[0168] Array number 61 N692A / N803D mutant SpyCas9 AA sequence.

[0169]

Chemical Structure

[0170] Array number 62 R691A / S845A mutant SpyCas9 AA sequence.

[0171]

Chemical Structure

[0172] Array number 63 N692A / S845A mutant SpyCas9 AA sequence.

[0173] [Chemical formula]

[0174] Sequence number 64 R691A / N854K mutant SpyCas9 AA sequence.

[0175] [Chemical formula]

[0176] Sequence number 65 N692A / N854K mutant SpyCas9 AA sequence.

[0177] [Chemical formula]

[0178] Sequence number 66 R691A / S872A mutant SpyCas9 AA sequence.

[0179] [Chemical formula]

[0180] Sequence number 67 N692A / S872A mutant SpyCas9 AA sequence.

[0181] [Chemical formula]

[0182] Sequence number 68 R691A / N692A / T740A mutant SpyCas9 AA sequence. <s

[0183] [Chemical formula]

[0184] Sequence ID 69 R691A / N692A / S845A mutant SpyCas9 AA sequence.

[0185] [ka]

[0186] Sequence ID 70 R691A / N692A / S872A variant SpyCas9 AA sequence.

[0187] [ka]

[0188] Sequence ID 71 R691S mutant SpyCas9 AA sequence.

[0189] [ka]

[0190] Sequence ID 72 R691N mutant SpyCas9 AA sequence.

[0191] [ka]

[0192] Sequence ID 73 R691D mutant SpyCas9 AA sequence.

[0193] [ka]

[0194] Sequence ID 74 R691C mutant SpyCas9 AA sequence.

[0195] [ka]

[0196] Sequence ID 75 R691Q mutation SpyCas9 AA sequence.

[0197] [ka]

[0198] Sequence ID 76 R691E mutant SpyCas9 AA sequence.

[0199] [ka]

[0200] Sequence ID 77 R691G mutant SpyCas9 AA sequence.

[0201] [ka]

[0202] Sequence ID 78 R691H mutant SpyCas9 AA sequence.

[0203] [ka]

[0204] Sequence ID 79 R691I mutant SpyCas9 AA sequence.

[0205] [ka]

[0206] Sequence ID 80 R691L mutant SpyCas9 AA sequence.

[0207] [ka]

[0208] Sequence ID 81 R691K mutant SpyCas9 AA sequence.

[0209] [ka]

[0210] Sequence ID 82 R691M mutant SpyCas9 AA sequence.

[0211] [ka]

[0212] Sequence ID 83 R691F mutant SpyCas9 AA sequence.

[0213] [ka]

[0214] Sequence ID 84 R691P mutant SpyCas9 AA sequence.

[0215] [ka]

[0216] Sequence ID 85 R691T mutant SpyCas9 AA sequence.

[0217] [ka]

[0218] Sequence ID 86 R691W mutant SpyCas9 AA sequence.

[0219] [ka]

[0220] Sequence ID 87 R691Y mutant SpyCas9 AA sequence.

[0221] [ka]

[0222] Sequence ID 88 R691V mutant SpyCas9 AA sequence.

[0223] [ka]

[0224] [Example 2] Bacterial gene screening to enrich mutant Cas9 peptides that maintain high on-target activity while reducing off-target cleavage. The following examples detail a gene screening performed in E. coli to identify candidate Cas9 mutant enzymes of interest from a library of approximately 250,000 mutant clones for later, more detailed characterization.

[0225] All Cas9 variants with reduced off-target effects, as revealed in conventional techniques, were developed using rational design methods based on previously published crystalline and co-crystal structures of Cas9, Cas9-gRNA, and Cas9-gRNA-DNA complexes, by creating alanine substitutions of charged amino acids adjacent to the Cas9 nucleic acid binding pocket. This restricts the sequence space available for mutation to a very limited number of amino acid residues. The present invention identifies novel useful Cas9 variants by using, instead of the above, an unbiased screening of a large number of random variants generated by low-fidelity PCR of Cas9 expression cassettes, which significantly expands the potential sequence space investigated for useful variants.

[0226] In this invention, the inventors selected any amino acid substitution in Cas9 that facilitates on-target cleavage but avoids off-target cleavage using a bacterial screening method. The screening method was adapted from screenings previously performed for other applications. See Chen and Zhao (Nucleic Acids Research, 33(18)pe154, 2005) and Kleinstiver et al. (Nature, 523, pp. 481-485, 2015). This screening is based on co-transformation of E. coli cells using two plasmids: i) a toxin plasmid encoding an arabinose-inducible cell proliferation toxin linked to a CRISPR / Cas9 on-target cleavage site (VEGFA3, HEKSite4, or EMX1, SEQ ID NOs. 133, 135, 137), where on-target cleavage eliminates toxin production (i.e., if the on-target site is not cleaved, the cells die); and ii) a chloramphenicol resistance plasmid containing a randomly mutagenerated (approximately 6 mutations per kilobase) Cas9 sequence, a single guide RNA (sgRNA) specific to each on-target site, and known off-target cleavage sites for each guide RNA (SEQ ID NOs. 134, 136, 138) (if cleavage occurs, the resistance gene is not expressed, and the cells are linked to chloramphenicol expression so that they die when exposed to the selection marker chloramphenicol). The screening design allows for the sequential use of different on-target sites (toxins) paired with appropriate off-target sites (chloramphenicol), thereby enabling the screening to be repeated to ensure that isolates were not selected based solely on their performance against a single gRNA target site.

[0227] The screening method was as follows: E. coli K12 strain MG1655 was transformed with a toxin plasmid containing the VEGF A3 target site in the absence of arabinose. In this case, no toxin was produced, and cell viability was possible. Cells with stably replicating toxin plasmids were then transformed with a chloramphenicol Cas9-sgRNA off-target plasmid, grown nonselectively at 37°C for 1 hour, and harvested. The transformed cells were then seeded on selective medium containing both chloramphenicol and arabinose. The grown bacteria were: i) bacteria that successfully transformed with the Cas9-sgRNA off-target plasmid, ii) bacteria that expressed enough Cas9 and VEGFA3 sgRNA to cleave the toxin-on-target plasmid, and iii) bacteria that avoided cleavage of the chloramphenicol Cas9-sgRNA off-target plasmid to enable sufficient chloramphenicol resistance for selective survival. A pool of candidate mutations that allowed viability was generated by all three tested guides (VEGFA3, HEKSite4, and EMX1). Within this pool, mutations isolated multiple times through the screening process (a total of 94 clones) were further analyzed in mammalian cells. A schematic of this screening method is shown in Figure 1.

[0228] [Example 3] Plasmid delivery of novel Cas9 mutants reduces off-target genes while maintaining on-target activity.

[0229] The following examples demonstrate the present invention's ability to reduce off-target gene editing activity by plasmid delivery of the Cas9 nuclease. A single point mutation identified in primary screening (Example 2) was given as a starting point, and selected double or triple mutant variants were generated from this starting point by site-directed mutagenesis. In plasmid expression settings where the Cas9 enzyme is overexpressed, clones with combinations of multiple mutations were able to limit the effect of on-target editing activity and show an improved reduction in off-target editing activity.

[0230] Alt-R® SpCas9 expression plasmids (WT) were modified by site-directed mutagenesis, and the presence of the indicated mutations was confirmed by DNA sequencing. CRISPR / Cas9 experiments were performed using two sets of Alt-R® crRNA and tracrRNA systems that were simultaneously co-transfected with different WT and mutant Alt-R® Cas9 expression plasmids. Alt-R® crRNA targeting NGG PAM-containing sequences at the EMX1 and HEKSite4 loci (Table 1, SEQ ID NOs. 113 and 114) was double-stranded into Alt-R® tracrRNA at a 1:1 molar ratio (3 μM) by heating at 95°C for 5 minutes and then slowly cooling to 25°C. Triple reverse transfection was performed using 0.5 μl of Transit-X2 (Mirus Bio LLC), a 30 nM EMX1 or HEKSite4 Alt-R® gRNA complex, and 0.1 μg of Alt-R® Cas9 plasmid (containing either WT or the indicated mutant). Transfection lipid complexes were formed at room temperature for 20 minutes according to the manufacturer's instructions, and 40,000 HEK293 cells were added to each transfection. After incubation at 37°C and 5% CO2 for 48 hours, adherent cells were washed with 0.1 ml of PBS and lysed with 0.05 ml of QuickExtract® DNA extraction solution. The cell lysates were incubated at 65°C for 15 minutes, followed by thermal inactivation at 98°C for 3 minutes. The crude DNA sample was then diluted 3-fold with 0.1 ml of ddH2O and used as a PCR template. The PCR primers and expected T7 endonuclease 1 (T7E1) digestion patterns are shown in Table 1 (SEQ ID NOs: 121-128). KAPA HiFi DNA polymerase and the following cycle parameters: 95 5:00 , (98 0:20 , 64 0:15 , 72 0:30 ) 29 repetitions, 72 2:00PCR was used to amplify a ≦1 kb fragment of either the EMX1 or HEKSite4 locus using the following cycling parameters: 95 cooled to 85 over 1 minute 10:00 85 cooled to 75 over 1 minute 1:00 75 cooled to 65 over 1 minute 1:00 65 cooled to 55 over 1 minute 1:00 55 cooled to 45 over 1 minute 1:00 45 cooled to 35 over 1 minute 1:00 35 cooled to 25 over 1 minute 1:00 25 1:00 was used to form heteroduplexes. X Y The aforementioned numbers described as X represent the isothermal temperature (in Celsius) and Y represents the time period (represented as "n:00" for minutes or "0:nn" for seconds, where n is an integer). Heteroduplexes were cleaved by adding 2 U of T7 endonuclease I (New England Biolabs) at 37 °C and incubating for 1 hour, and the cleavage products were analyzed by capillary electrophoresis (Fragment Analyzer, Advanced Analytical). The T7E1 mismatch cleavage assay was utilized to evaluate DNA editing efficiency in this and subsequent examples. The complete protocol is described (see Jacobi et al., Methods, 121 - 122, pp. 16 - 28, 2017).

[0231] These results indicate that point mutations at the indicated Cas9 amino acid positions (SEQ ID NOs: 9, 11, 13, 15, 16, 17, 18, 20, 22, 23, 25, 27, 28, 29, 30, 31, 33, 34, 36, 37, and 71) reduce off-target gene editing activity for crRNAs targeting EMX1 (Figure 2) or the HEKSite4 locus (Figure 3). Many of the point mutations isolated from our screening resulted in substitutions of amino acids other than alanine. For these variants from the initial screening, which were non-alanine substitutions, we performed site-directed mutagenesis to change these mutations to alanine substitutions at these positions and compared them to both WT Cas9 and the original amino acid substitutions (new alanine variants include SEQ ID NOs: 7, 10, 12, 14, 19, 21, 24, 26, 32, 35, and 38). The inventors observed similar phenotypes between the original mutants isolated from primary screening and novel alanine mutants (Figures 4 and 5). In some cases, alanine substitution resulted in higher on-target editing activity and lower off-target editing activity. Generally, the alanine mutants functioned similarly to or better than the isolated original mutants, and therefore the forward mutation combinations were tested only using alanine substitutions. Mutations at positions R691 and N692 showed the greatest decrease in off-target gene editing activity for both EMX1 and HEKSite4 crRNAs. Mutations at R691 demonstrated on-target editing activity at WT Cas9 levels, while mutations at N692 showed a slight decrease in on-target editing activity for both crRNAs. For this reason, the inventors chose to create double and triple mutant combinations based on either the R691A or N692A mutation as a starting point. The inventors also created and tested the R691A / N692A double mutant. The new double mutant Cas9 sequences include sequence numbers 39-70. All combinations of these mutations had an additive effect in that no detectable off-target editing activity was detected (Figure 6).Two previously published high-fidelity Cas9 proteins, eSpCas9(1.1)(K848A, K1003A, and R1060A) (Slaymaker et al., Science, 351, pp. 84-88, 2016) and SpCas9-HF1(N497A, R661A, Q695A, and Q926A) (Kleinstiver et al., Nature, pp. 490-495, 2016), also exhibited undetectable off-target editing activity when delivered by plasmid, but reduced on-target editing activity compared to the wild-type protein. The variants of the present invention, when delivered as plasmids, have a superior overall editing activity profile compared to these variants from the prior art. Table 1 provides the sequences of the target-specific protospacer domains of all CRISPR gRNAs used in the examples. These RNA sequences represent the variable domains of the gRNAs that change with the target site. In practice, the protospacer domain is adjacent to further universal RNA sequences to contain a complete functional Cas9 crRNA or sgRNA (see Jinek et al., Science, 2012, 337(6096): pp. 816-821 and Jacobi et al., Methods, 2017, pp. 121-122, 16-28). Table 2 shows the sequences of the DNA on-target and off-target domains and dsDNA GUIDE-Seq tags studied in the examples.

[0232] [Table 1] JPEG2026065115000095.jpg11274 JPEG2026065115000096.jpg4175

[0233] [Table 2]

[0234] [Example 4] RNP delivery by novel Cas9 mutants reduces off-target editing activity while maintaining on-target editing activity.

[0235] The following examples demonstrate the ability of the Cas9 variant of the present invention to reduce off-target gene editing activity and maintain on-target editing activity when the Cas9-gRNA complex is delivered to mammalian cells as an RNP complex.

[0236] The Cas9 amino acid mutations described in this invention were transferred to the environment of a Cas9 protein expression / purification plasmid enabling recombinant protein expression in E. coli. The resulting protein contains an NLS domain to facilitate nuclear delivery in mammalian cells and an HIS tag to simplify purification (see WT Cas9 DNA sequence, SEQ ID NO: 1 and R691A mutant, SEQ ID NO: 3). The amino acid sequence with domain addition is shown as an exemplary model (WT SEQ ID NO: 6, R691A mutant SEQ ID NO: 8). Wild-type and mutant Cas9 proteins were purified by immobilized metal affinity and heparin chromatography. The publicly available high-fidelity Cas9 proteins, eSpCas9(1.1) and SpCas9-HF1, were also purified using this method. CRISPR / Cas9 experiments were performed by first forming a 1 μM RNP complex with the purified Cas9 protein and 2 parts Alt-R® RNA (crRNA:tracrRNA complex) in Opti-MEM for 5 minutes at 25°C. Alt-R® crRNA targeted the HPRT gene (SEQ ID NOs. 89-100) and the CTLA4 gene (SEQ ID NOs. 101-112) and was delivered to HEK293 cells (40,000 cells / well) by reverse transfection of a pre-formed RNP complex using 1.2 μl of RNAiMAX. The experiment was performed in a biological triplicate, and cells were lysed after 48 hours of incubation at 37°C and 5% CO2. DNA extraction, PCR amplification, and T7E1 digestion were performed as described for the plasmid-based experiment in Example 3. PCR amplification primers are listed in Table 1 (SEQ ID NOs. 117-122).

[0237] Figure 7 shows the genome editing efficiency using the RNP method in mammalian cells at 12 sites of the human HPRT gene, comparing WT, mutant R691A, eSpCas9(1.1), and Cas9-HF1 proteins. Figure 8 shows the efficiency for 12 sites of the human CTLA4 gene. The R691A Cas9 mutant of the present invention showed on-target editing activity equivalent to wild-type Cas9 at 95% of the tested sites, while SpCas9-HF1 and eSpCas9(1.1) showed useful function at only 29% and 57% of the sites, respectively.

[0238] The same system was used to study off-target editing activity at previously identified crRNA sites in three different genes (EMX1 SEQ ID NO: 113, HEKSite4 SEQ ID NO: 114, and VEGFA3 SEQ ID NO: 116). On-target sites are shown alongside off-target sites (SEQ ID NOs: 121-126, 129, and 130) in Figure 9. Significant off-target editing activity was observed at all three sites for the WT Cas9 enzyme, but none of the three mutant Cas9 enzymes showed off-target activity detectable using this assay. However, the novel R691A mutant of the present invention showed identical on-target activity at these three sites, while only the conventional mutants eSpCas9(1.1) and Cas9-HF1 showed full activity at the EMX1 site, and significantly reduced activity was observed at the HEKSite4 and VEGF3A loci. This demonstrates the usefulness of the present invention: the R691A Cas9 mutant maintains high on-target activity while reducing off-target activity, providing a significant improvement over the performance of existing mutants from the prior art.

[0239] Further Cas9 variants (including single, double, and triple variants) were prepared as purified recombinant proteins, and their on-target activity at site HPRT-38509 (SEQ ID NO: 92) (Figure 10) and on-target versus off-target activity at HEKSite4 (SEQ ID NO: 114) were studied using RNP delivery in mammalian cells as described above. The Cas9 enzymes studied included the WT (SEQ ID NO: 5) and variants (SEQ ID NOs: 7, 19, 22, 33, 36, 52, 62, 66, 68, 69, and 70). HPRT 38509 is a gRNA site that typically does not exhibit high levels of editing activity and is sensitive to changes in Cas9 activity.

[0240] Figure 10 shows that all of the tested Cas9 single mutants exhibited high levels of on-target activity, while all of the double and triple mutants showed significant loss of activity when used in mammalian cells with the RNP method. Note that these identical double mutants showed good activity when overexpressed from plasmid templates (Figure 6).

[0241] These mutants were also delivered as RNPs to test for off-target activity and the HEKSite4 locus. Overall, the N692A mutant functioned similarly to R691A, with only a slight reduction in on-target editing activity (Figure 10) and undetectable off-target editing activity (Figure 11). Other single mutants in the isolate showed excellent on-target editing activity (Figure 10), but with only slight or no reduction in off-target editing activity at this challenging site (Figure 11). Multiple combinations of mutants containing R691A or N692A showed excellent on-target editing activity coupled with undetectable off-target editing activity when delivered using plasmids, but these mutants showed reduced on-target editing activity when delivered using RNPs (Figures 10 and 11). The R691A mutant showed the best overall combination of reduced off-target editing activity and maintained on-target editing activity at multiple sites tested, so this site was studied in more depth.

[0242] [Example 5] Further testing of amino acid mutations at position R691 using plasmid and RNP delivery methods. Previously, site R691 has been characterized as WT and as mutants R691A and R691S. This example demonstrates the activity of 17 other possible amino acid substitutions at this position in Cas9.

[0243] Seventeen new amino acid substitutions at this site were introduced into mammalian Cas9 expression plasmids using site-directed mutagenesis, and their function in HEK293 cells was tested using plasmid delivery (method described in Example 3). On-target editing activity was studied using crRNA HPRT 38509 (SEQ ID NO: 92), and the results for all 20 possible amino acids at this site (SEQ ID NOs: 5, 7, and 71-88) are shown in Figure 12. Mutants R691N, R691C, R691T, R691I, R691L, and R691V showed reduced on-target activity, while all WT and mutants utilizing 14 other amino acids at this site showed high activity.

[0244] The combinations of on-target and off-target activity for this set of 20 Cas9 variants were studied at crRNA HEKSite4 (SEQ ID NO: 114), a highly active site for both on-target and off-target activity in WT Cas9. Figure 13 demonstrates that WT Cas9, as well as mutants R691K and R691P, showed high off-target activity at this site, while all other 17 Cas9 mutants showed significantly reduced off-target activity. This demonstrates that site R691 is an ideal site for mutation to improve Cas9 function, and that various different amino acid substitutions function well in this environment.

[0245] The best-performing recombinant proteins of these mutants, R691D, R691G, R691H, R691Y, and R691W, were prepared and tested for on-target and off-target editing activity compared to WT Cas9 and mutant R691A using RNP delivery (method described in Example 4). As shown in Figures 14 and 15, all of these mutants showed very similar levels of on-target editing activity at crRNA sites HPRT 38509 (SEQ ID NO: 92), HPRT 38087 (SEQ ID NO: 94), EMX1 (SEQ ID NO: 113), and HEKSite4 (SEQ ID NO: 114). All mutants also showed a significant decrease in off-target activity at sites EMX1 and HEKSite4 (Figure 15), although low but detectable off-target editing activity was observed for the R691G, R691H, and R691Y mutants. The R691A, R691D, and R691W variants provided the best combination of reduced on-target and off-target editing activity when both plasmid and RNP delivery methods were considered.

[0246] The R691A mutant was tested for overall off-target effects using a publicly available, unbiased genome-wide next-generation sequencing (NGS) assay called Guide-Seq (Tsai et al., Nature Biotechnology, 33, pp. 187-197, 2015). This was a source of validated off-target sites for the EMX1, HEKSite4, and VEGF3A cRNAs studied in the above examples. The Guide-Seq protocol was performed as recommended, using either WT Cas9 or R691A mutant Cas9 with RNP delivery, and employing crRNA guides EMX1 (SEQ ID NO: 113), VEGFA3 (SEQ ID NO: 116), and AR (SEQ ID NO: 115). NGS library construction and data processing were carried out as previously described (Tsai et al., Nature Biotechnology 33: pp. 187-197, 2015), and the results (Figure 16) demonstrate that the R691A mutant significantly reduces overall off-target editing activity while maintaining on-target editing activity compared to the WT Cas9 nuclease.

[0247] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent that each reference is incorporated by reference individually and specifically as is indicated and as is contained herein in whole.

[0248] In the context describing the present invention (particularly in the context of the following claims), the terms “a,” “an,” and “the,” as well as similar demonstrative pronouns, are to be interpreted as including both singular and plural unless otherwise indicated herein or unless clearly contradicted by the context. The terms “comprising,” “having,” “including,” and “containing” are to be interpreted as open-ended terms unless otherwise indicated herein (i.e., “including, but not limited to”). The enumeration of value ranges herein is intended merely as a convenient way of referring individually to each distinct value contained within that range unless otherwise indicated herein, and each distinct value is incorporated herein as if it were individually enumerated herein. All methods described herein may be carried out in any suitable order unless otherwise indicated herein or unless clearly contradicted by the context. Any examples or illustrative words (e.g., "like") provided herein are intended only to better illustrate the invention and, unless otherwise requested, do not limit the scope of the invention. No word or element provided herein should be construed as indicating that any element not claimed is essential for the practice of the invention.

[0249] Preferred embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will appropriately use such variations, and the inventors intend that the Invention may be carried out in ways different from those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all these possible variations is incorporated into the Invention unless otherwise indicated herein or unless it is clearly inconsistent with the context.

Claims

1. An isolated mutant Cas9 protein that is active in the CRISPR / Cas endonuclease system, wherein the CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

2. (a) A single substitution mutation introduced into the WT-Cas9 protein, selected from the positions R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925, or (b) Double substitution mutations introduced into the WT-Cas9 protein, selected from two of the following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872, and R925. The isolated mutant Cas9 protein according to claim 1, comprising a substitution mutation selected from the group consisting of the following.

3. The isolated mutant Cas9 protein according to claim 1, comprising a substitution mutation selected from the group consisting of double substitution mutations introduced into the WT-Cas9 protein, selected from two of the following positions: R494C or R494A; N522K or N522A; N588D or N588A; N612A; T657A; S663A; R691S or R691A; N692D or N692A; S730G or S730A; T740A; R765G or R765A; T770K or T770A; N776A; R778A; R783A; S793A; N803D or N803A; S845A; N854K or N854A; S872A; and R925C or R925A.

4. An isolated mutant Cas9 protein according to claim 1, selected from the group consisting of SEQ ID NOs: 7 to 38.

5. An isolated mutant Cas9 protein according to claim 1, selected from the group consisting of SEQ ID NOs: 39 to 88.

6. Mutant Cas9 protein, and gRNA complex An isolated ribonucleoprotein complex comprising, The isolated ribonucleoprotein complex is active as a CRISPR / Cas endonuclease system, and the resulting CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

7. The isolated ribonucleoprotein complex according to claim 6, wherein the gRNA comprises crRNA and tracrRNA in a stoichiometric (1:1) ratio.

8. gRNA, Isolated crRNA containing Alt-R® crRNA for a specific editing target site for a given gene locus, and Alt-R (trademark) tracrRNA containing tracrRNA The isolated ribonucleoprotein complex according to claim 6, comprising

9. The isolated ribonucleoprotein complex according to claim 6, wherein the gRNA contains sgRNA.

10. Mutant Cas9 protein, (a) A single substitution mutation introduced into the WT-Cas9 protein, selected from the positions R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925, or (b) Double substitution mutations introduced into the WT-Cas9 protein, selected from two of the following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872, and R925. The isolated ribonucleoprotein complex according to claim 6, comprising a substitution mutation selected from the group consisting of the following.

11. The isolated ribonucleoprotein complex according to claim 6, wherein the mutant Cas9 protein comprises substitution mutations selected from the group consisting of double substitution mutations introduced into the WT-Cas9 protein, selected from two positions selected from among R494C or R494A; N522K or N522A; N588D or N588A; N612A; T657A; S663A; R691S or R691A; N692D or N692A; S730G or S730A; T740A; R765G or R765A; T770K or T770A; N776A; R778A; R783A; S793A; N803D or N803A; S845A; N854K or N854A; S872A; and R925C or R925A.

12. The isolated ribonucleoprotein complex according to claim 6, wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 7 to 38.

13. The isolated ribonucleoprotein complex according to claim 6, wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 39 to 88.

14. An isolated nucleic acid encoding a mutant Cas9 protein, wherein the mutant Cas9 protein is active in the CRISPR / Cas endonuclease system, and the CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system.

15. Mutant Cas9 protein, (a) A single substitution mutation introduced into the WT-Cas9 protein, selected from the positions R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925, or (b) an isolated nucleic acid encoding the mutant Cas9 protein according to claim 14, comprising a substitution mutation selected from the group consisting of double substitution mutations introduced into the WT-Cas9 protein, selected from two of the positions of R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925.

16. Isolated nucleic acids encoding the mutant Cas9 protein according to claim 14, wherein the mutant Cas9 protein comprises substitution mutations selected from the group consisting of double substitution mutations introduced into the WT-Cas9 protein, selected from two positions selected from among R494C or R494A; N522K or N522A; N588D or N588A; N612A; T657A; S663A; R691S or R691A; N692D or N692A; S730G or S730A; T740A; R765G or R765A; T770K or T770A; N776A; R778A; R783A; S793A; N803D or N803A; S845A; N854K or N854A; S872A; and R925C or R925A.

17. An isolated nucleic acid encoding a mutant Cas9 protein according to claim 14, wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 7 to 38.

18. An isolated nucleic acid encoding a mutant Cas9 protein according to claim 14, wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 39 to 88.

19. A CRISPR / Cas endonuclease system containing mutant Cas9 protein and gRNA, which exhibits reduced off-target editing activity and maintained on-target editing activity compared to a wild-type CRISPR / Cas endonuclease system.

20. The CRISPR / Cas endonuclease system according to claim 19, encoded by a DNA expression vector.

21. The CRISPR / Cas endonuclease system according to claim 19, wherein the DNA expression vector includes a plasmid-based vector.

22. The CRISPR / Cas endonuclease system according to claim 19, wherein the DNA expression vector is selected from a bacterial expression vector and a eukaryotic cell expression vector.

23. The CRISPR / Cas endonuclease system according to claim 19, wherein the gRNA comprises crRNA and tracrRNA in a stoichiometric (1:1) ratio.

24. The CRISPR / Cas endonuclease system according to claim 23, wherein the crRNA comprises Alt-R® crRNA for a specific editing target site for a given gene locus, and the tracrRNA comprises Alt-R® tracrRNA.

25. The CRISPR / Cas endonuclease system according to claim 19, wherein the gRNA includes sgRNA.

26. Mutant Cas9 protein, (a) A single substitution mutation introduced into the WT-Cas9 protein, selected from the positions R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925, or (b) Double substitution mutations introduced into the WT-Cas9 protein, selected from two of the following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872, and R925. The CRISPR / Cas endonuclease system according to claim 19, comprising a substitution mutation selected from the group consisting of the following.

27. Mutant Cas9 proteins include R494C or R494A; N522K or N522A; N588D or N588A; N612A; T657A; S663A; R691S or R691A; N692D or N692A; S730G or S730A; T740A; R765G or R765A; T770K or T770A; N776A; R778A; R The CRISPR / Cas endonuclease system according to claim 19, comprising substitution mutations selected from the group consisting of double substitution mutations introduced into the WT-Cas9 protein, selected from two of the following positions: 783A; S793A; N803D or N803A; S845A; N854K or N854A; S872A; and R925C or R925A.

28. The CRISPR / Cas endonuclease system according to claim 19, wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 7 to 38.

29. The CRISPR / Cas endonuclease system according to claim 19, wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 39 to 88.

30. A method for performing gene editing that reduces off-target editing activity and / or increases on-target editing activity, A method comprising the step of contacting a candidate editing target site locus with an active CRISPR / Cas endonuclease system having a mutant Cas9 protein, wherein the active CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to a wild-type CRISPR / Cas endonuclease system.

31. Mutant Cas9 protein, (a) A single substitution mutation introduced into the WT-Cas9 protein, selected from the positions R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925 or (b) Double substitution mutations introduced into the WT-Cas9 protein, selected from two of the following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872, and R925. The method according to claim 30, comprising a substitution mutation selected from the group consisting of the following.

32. WT-Cas9 protein in which the mutant Cas9 protein is selected from two of the following positions: R494C or R494A; N522K or N522A; N588D or N588A; N612A; T657A; S663A; R691A; N692D or N692A; S730G or S730A; T740A; R765G or R765A; T770K or T770A; N776A; R778A; R783A; S793A; N803D or N803A; S845A; N854K or N854A; S872A; and R925C or R925A. The method according to claim 30, comprising a substitution mutation selected from the group consisting of double substitution mutations introduced into the .

33. The method according to claim 30, wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 7 to 38.

34. The method according to claim 30, wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 39 to 88.

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