S. pyogenes cas9 mutant genes and polypeptides encoded by the same

Abstract

To provide mutant Cas9 nucleic acids and proteins for use in CRISPR / Cas endonuclease systems, and methods of using them.SOLUTION: The invention provides an isolated mutant Cas9 protein, where the isolated mutant Cas9 protein is active in a CRISPR / Cas endonuclease system, where the CRISPR / Cas endonuclease system displays reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR / Cas endonuclease system. The invention also includes isolated nucleic acids encoding mutant Cas9 proteins, ribonucleoprotein complexes and CRSPR / Cas endonuclease systems having mutant Cas9 proteins that display reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR / Cas endonuclease system.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application claims the benefit of priority under 35 U.S.C. §119 to U.S. Provisional Patent Application No. 62 / 405,601, filed October 7, 2016, entitled "NOVEL S.PYOGENES CAS9 MUTATIONS THAT REDUCE OFF TARGET GENE EDITING WHILE MAINTAINING ON TARGET POTENCY," the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is hereby incorporated 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 THEINVENTION The present invention relates to Cas9 mutant genes, polypeptides encoded thereby and their use in compositions of CRISPR-Cas systems. [Background technology]

[0004] The use of clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas proteins (CRISPR-Cas systems) for site-specific DNA cleavage has shown great potential for many biological applications. CRISPR has been used for genome editing, genome-scale specific targeting of transcriptional repressors (CRISPRi) and activators (CRISPRa) to endogenous genes, and other applications of RNA-guided DNA targeting by Cas enzymes.

[0005] CRISPR-Cas systems are native to bacteria and archaea and provide adaptive immunity against viruses and plasmids. Three classes of CRISPR-Cas systems can potentially be adapted for research and therapeutic reagents. Type II CRISPR systems have desirable features in utilizing a single CRISPR-associated (Cas) nuclease (specifically, Cas9) in complex with an appropriate guide RNA (gRNA). In bacteria or archaea, the Cas9 guide RNA comprises two separate RNA species. A target-specific CRISPR-activating RNA (crRNA) binds to and guides the Cas9 / gRNA complex to target a specific DNA sequence. The crRNA has two functional domains, a 5' domain that is target-specific and a 3' domain that guides the binding of the crRNA to the trans-activating crRNA (tracrRNA). The tracrRNA is a longer universal RNA that binds to the crRNA and mediates the binding of the gRNA complex to Cas9. Binding of the tracrRNA induces a change in Cas9 structure, shifting it from an inactive to an active conformation. The gRNA function can also be provided as an artificial single guide RNA (sgRNA) in which the crRNA and tracrRNA are fused into a single species (see Jinek, M. et al., Science 337, 816-21, 2012). The sgRNA format allows for transcription of a functional gRNA from a single transcription unit, which can be provided by a double-stranded DNA (dsDNA) cassette containing a transcription promoter and the sgRNA sequence. In mammalian systems, these RNAs have been introduced by transfection of a DNA cassette containing an RNA Pol III promoter (such as U6 or H1) driving RNA transcription, viral vectors, and single-stranded RNA after in vitro transcription (see Xu, T. et al., Appl Environ Microbiol, 2014. 80(5):1544-52).

[0006] In the CRISPR-Cas system, using as an example the system present in Streptococcus pyogenes (S.py. or Spy), the native crRNA is about 42 bases long and contains a 5' region of about 20 bases 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, typically about 22 bases long, that is complementary to a region of the tracrRNA sequence and mediates binding of the crRNA to the tracrRNA. The crRNA:tracrRNA complex contains a functional gRNA that can direct Cas9 cleavage of a complementary target DNA. The native tracrRNA has a 5' region that is about 85-90 bases long and contains 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 binding of the crRNA:tracrRNA complex to Cas9.

[0007] Jinek et al. extensively investigated the physical domains of crRNA and tracrRNA required for proper functioning of the CRISPR-Cas system (Science, 2012, 337(6096):816-21). They devised truncated crRNA:tracrRNA fragments that could still function in CRISPR-Cas, where the crRNA was wild-type 42 nucleotides and the tracrRNA was truncated to 75 nucleotides. They also developed an embodiment in which the crRNA and tracrRNA were linked with 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 solved the crystal structure of Streptococcus pyogenes Cas9 (SpyCas9). In Jinek, M et al., the structure did not show the nuclease in complex with either the guide RNA or the target DNA. They performed molecular modeling experiments to reveal predicted interactions between the proteins in the RNA and DNA complex (Science, 2014, 343, 1215, DOI:10.1126 / science / 1247997).

[0009] In Nishimasu, H et al., the crystal structure of Spy Cas9 has been shown at 2.5 angstrom resolution in complex with sgRNA and its target DNA (Cell, 2014, 156(5):935-49, incorporated herein in its entirety). The crystal structure identified two lobes for the Cas9 enzyme: the recognition lobe (REC) and the nuclease lobe (NUC). The sgRNA:target DNA heteroduplex (negatively charged) lies in a positively charged groove between the two lobes. The REC lobe, which shows no structural similarity to known proteins and thus like the Cas9-specific functional domain, interacts with portions of the crRNA and tracrRNA that are complementary to each other.

[0010] Another group, Briner et al. (Mol Cell, 2014, 56(2):333-9, incorporated herein in its entirety) identified and characterized six conserved modules within the natural crRNA:tracrRNA duplex and sgRNA. Anders et al. (Nature, 2014, 513(7519), 569-73) elucidated the structural basis for DNA sequence recognition of protospacer associated motif (PAM) sequences by Cas9 in association with sgRNA guides.

[0011] The CRISPR-Cas endonuclease system is utilized in genome engineering as follows: the gRNA complex (either the 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 initiates unwinding of the target DNA, followed by annealing of the protospacer domain to the target, after which cleavage of the target DNA occurs. Cas9 contains two domains homologous to the endonucleases HNH and RuvC, respectively, the HNH domain cleaves the DNA strand complementary to the crRNA, and the RuvC-like domain cleaves the non-complementary strand. This results in a double-strand break in the genomic DNA. When repaired by non-homologous end joining (NHEJ), the break is typically repaired in an imprecise manner, resulting in a DNA sequence that is shifted by one or more bases, leading to disruption of the native DNA sequence, and often leading to frameshift mutations when this event occurs within a coding exon of a gene that codes for a protein.Break can also be repaired by homology directed recombination (HDR), which allows the insertion of new genetic material based on exogenous DNA introduced into the cell with the Cas9 / gRNA complex, which is introduced into the break site created by Cas9 cleavage.

[0012] Wild-type (WT) Cas9 protein cleaves most DNA targets with high efficiency, but exhibits a sufficient level of undesired off-target editing to complicate research applications and pose major concerns for medical applications. In this context, off-target cleavage is defined as a DNA cleavage event that occurs at a site where the genomic DNA target site is different from the perfect complementarity to the protospacer domain of the crRNA or sgRNA. It is undesirable to introduce a cleavage event at a non-targeted site via such an off-target cleavage pathway. Typically, cleavage is only desired at sites in the genome that have perfect complementarity to the gRNA. Several groups have published novel mutant Cas9 enzymes that exhibit reduced off-target cleavage activity (see Slaymaker et al., Science, 2016, 351, 84-88; Kleinstiver et al., Nature, 2016, 529, 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 mutation of specific amino acid residues in the Cas9 protein that 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. Although knowledge of the mechanism of action is not required to practice these inventions (i.e., to perform genome editing with improved specificity), it was initially thought that the improved fidelity mutants act by decreasing the relative affinity of the mutant Cas9 nuclease for the substrate DNA compared to the WT enzyme, making it more likely that mismatches between the guide RNA and the substrate DNA will destabilize. More recently, it has been proposed that the mutations limit the transition of the Cas9 structure from an inactive to an active conformation, and that this transition occurs less effectively in the presence of mismatches between the RNA guide and the DNA target. Regardless of the mechanism, these mutant Cas9 enzymes show reduced cleavage of target DNA with incomplete complementarity to the guide RNA, when necessary. However, this improved specificity comes at the cost of also reducing on-target activity, which is undesirable.In all three prior art examples disclosing Cas9 variants of improved specificity, genome editing using CRISPR / Cas9 technology was performed using plasmid or other expression-based approaches, i.e., methods first described in 2013 (see Cong et al., Science, 2013, 339, 819-823; Mali et al., Science, 2013, 339, 823-826). However, it is now understood that plasmid systems introduce complexities to genome editing. For example, plasmids can integrate into the host genome, thereby leading to other unwanted genomic changes or this can trigger an innate immune response, resulting in cell death. For these and other reasons, plasmid systems are not ideal for research applications where precise editing is desired, and are not practical for medical applications where such side effects may be unacceptable. More recently, methods using ribonucleoprotein (RNP) complexes in which recombinant Cas9 protein is precomplexed with synthetic gRNA have been shown to be preferable to using DNA-based expression constructs. RNP methods result in highly active genome editing with reduced side effects (see Cho et al., Genome Research, 2014, 24, 132-141; Aida et al., Genome Biology, 2015, 16, 87-98). It is therefore desirable to develop a high-fidelity genome editing method that is compatible with RNP protocols. All previously cited published examples describing Cas9 mutants of improved specificity utilized plasmid-based DNA expression cassettes to perform and study genome editing outcomes. This method results in high levels of overexpression of mutant Cas9 proteins for extended periods of time, increasing the apparent enzymatic activity of the mutants. The inventors demonstrate herein that these improved specificity Cas9 variants (eSpCas9(1.1) and Cas9-HF1) have reduced enzymatic activity when using the RNP method to perform genome editing, such that cleavage of the target DNA site is significantly impaired compared to cleavage by the WT Cas9 protein, such that in many cases target sites that act with high efficiency using the WT Cas9 protein do not show any evidence of cleavage using the mutant variants.The published mutant Cas9 proteins are therefore of limited utility for precise genome editing, especially when the more medically relevant RNP methods are utilized. [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, 84-88 [Non-Patent Document 9] Kleinstiver et al., Nature, 2016, 529, 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, 132-141 [Non-Patent Document 14] Aida et al., Genome Biology, 2015, 16, 87-98 Summary of the Invention [Problem to be solved by the invention]

[0014] Thus, there remains a need for methods to improve the specificity of Cas9 genome editing, particularly for variants of Cas9 that exhibit improved specificity while retaining high enzymatic activity similar to the WT Cas9 enzyme when utilized in an RNP format. [Means for solving the problem]

[0015] The present invention relates to Cas9 mutant genes and polypeptides for use in CRISPR systems and methods of use thereof.

[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 ("CRISPR / Cas endonuclease system"). The CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to wild-type CRISPR / Cas endonuclease system.

[0017] In a second aspect, an isolated ribonucleoprotein (RNP) complex is provided. The RNP complex comprises 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 shows reduced off-target editing activity and maintained on-target editing activity compared to wild-type CRISPR / Cas endonuclease system.

[0018] In a third aspect, there is provided an isolated nucleic acid encoding a mutant Cas9 protein, the mutant Cas9 protein being active in a CRISPR / Cas endonuclease system, the CRISPR / Cas endonuclease system exhibiting reduced off-target editing activity and maintained on-target editing activity compared to a wild-type CRISPR / Cas endonuclease system.

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

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

[0021] [Figure 1]FIG. 1 is a schematic diagram of the bacterial genetic screen utilized to select mutant Cas9 sequences with high on-target and low off-target cleavage activity. [Diagram 2] FIG. 1 shows exemplary single amino acid mutations in S. pyogenes Cas9 that reduce off-target editing when Cas9 nuclease is delivered by a plasmid (as described in Example 3). CRISPR / Cas9 editing experiments used a series of mutant Cas9 plasmid expression cassettes (expressing WT Cas9 or the indicated mutant Cas9) co-transfected with Alt-R® crRNA:tracrRNA complexes targeting the human EMX1 gene. Relative editing efficiencies were 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 define PAM sites in the genomic DNA target, and bold highlighted bases indicate base mismatches at the off-target site relative to the on-target site). Off-target sites were identified by Tsai et al. (Nature Biotechnology 33:187-197, 2015). Experimental conditions included 30 nM EMX1 crRNA:tracrRNA and 100 ng Cas9 plasmid introduced by lipofection with 0.5 microliters of Transit X-2 per well. Error bars represent standard error of the mean (n=3). [Diagram 3]FIG. 1 shows exemplary single amino acid mutations in S. pyogenes Cas9 that reduce off-target editing when Cas9 nuclease is delivered by a plasmid (as described in Example 3). CRISPR / Cas9 editing experiments used a series of mutant Cas9 plasmid expression cassettes (expressing WT Cas9 or the indicated mutant Cas9) co-transfected with Alt-R® crRNA:tracrRNA complexes targeting the human HEK Site4 locus. Relative editing efficiencies were 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 define PAM sites in the genomic DNA target, and bold highlighted bases indicate base mismatches at the off-target site relative to the on-target site). Off-target sites were identified by Tsai et al. (Nature Biotechnology 33:187-197, 2015). Experimental conditions included 30 nM EMX1 crRNA:tracrRNA and 100 ng Cas9 plasmid introduced by lipofection with 0.5 microliters of Transit X-2 per well. Error bars represent standard error of the mean (n=3). [Figure 4]FIG. 2 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 alternatively replaced by alanine. Cas9 gRNA complexes are delivered as shown in FIG. 2 using an Alt-R® crRNA:tracrRNA complex targeting the EMX1 locus. 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), where underlined nucleotides clarify the PAM site in the genomic DNA target, and bases highlighted in bold indicate base mismatches of the off-target site relative to the on-target site). In each case, the bacterially selected mutant and its alanine-substituted counterpart are aligned. The experimental conditions and error analysis were identical to those shown in FIG. [Diagram 5]FIG. 3 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 alternatively replaced by alanine. Cas9 gRNA complexes are delivered as shown in FIG. 3 using an Alt-R® crRNA:tracrRNA complex targeting the HEKSite4 locus. Relative on-target editing efficiency is shown by a black bar on the left side of the Y-axis (5'-GGCACTGCGGCTGGAGGTGGGGG-3' (SEQ ID NO: 135)), and editing at a known off-target site is shown by a white bar on the right side of the Y-axis (off-target sequence 5'-GGCACGACGGCTGGAGGTGGGGG-3' (SEQ ID NO: 136), underlined nucleotides clarify the PAM site in the genomic DNA target, and bases highlighted in bold indicate base mismatches of the off-target site relative to the on-target site). In each case, the bacterially selected mutant and its alanine-substituted counterpart are aligned. The experimental conditions and error analysis were identical to those shown in FIG. [Figure 6]FIG. 1 shows an exemplary CRISPR / Cas9 editing experiment showing relative on-target and off-target editing efficiencies comparing different single and double amino acid mutations in the Cas9 protein, where the Cas9 gRNA complex is delivered as shown in FIG. 3 with the Alt-R® crRNA:tracrRNA complex targeting the HEKSite4 locus. The 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), underlined nucleotides clarify the PAM site in the genomic DNA target, and the bold highlighted bases indicate base mismatches of the off-target site relative to the on-target site). The experimental conditions and error analysis are identical to those shown in FIGS. 2 and 3 and 4 and 5. [Figure 7] FIG. 1 demonstrates that purified recombinant R691A Cas9 mutant protein, when delivered as a ribonucleoprotein (RNP) complex, exhibits superior on-target editing activity to known eSpCas9(1.1) or SpCas9-HF1 proteins. RNP complexes were formed (1 μM) 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. RNP delivery to cells included 10 nM RNP (1:1:1 ratio of Cas9:tracrRNA:crRNA (Alt-R crRNA)) with 1.2 microliters of RNAiMAX lipofection in HEK293 cells 48 hours prior to analysis. Error bars represent standard error of the mean. [Figure 8]FIG. 7 demonstrates that purified recombinant R691A Cas9 mutant protein exhibits superior on-target editing activity to known eSpCas9(1.1) or SpCas9-HF1 proteins when delivered as a ribonucleoprotein (RNP) complex. RNP complexes were formed (1 μM) with WT (black), eSpCas9(1.1) (white), SpCas9-HF1 (grey) 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. Experimental details and error analysis are similar to those described in FIG. 7. [Figure 9] Figure 1 shows exemplary results of an experiment demonstrating that off-target editing activity is undetectable by T7EI analysis with R691A mutant Cas9 when delivered as an RNP complex at three independent, validated off-target sites for three different crRNAs. RNP complexes were formed (1 μM) with WT (black), eSpCas9(1.1) (white), SpCas9-HF1 (grey) or R691A (hatched) Cas9 proteins and Alt-R crRNA:tracrRNA gRNA complexes targeting human EMX1 (left) (SEQ ID NO: 113), HEKSite4 (center) (SEQ ID NO: 114) or VEGFA3 (right) (SEQ ID NO: 116) loci. On-target and off-target sites for EMX1 and HEKSite4 loci are as described in Figures 2 and 3, respectively. On-target editing for the VEGFA3 locus (5'-GGTGAGTGAGTGTGTGCGTGTGG-3' (SEQ ID NO: 137)) and known problematic off-target sites for this guide are shown (5'-AGTGAGTGAGTGTGTGTGTGGGG-3' (SEQ ID NO: 138), underlined nucleotides indicate PAM sites and base differences between the off-target and on-target sites are highlighted in bold font (Tsai et al., Nature Biotechnology 33:187-197, 2015). [Figure 10]FIG. 1 shows exemplary results of experiments demonstrating that while the R691A mutant maintains highly on-target editing active guide sites that routinely demonstrate low efficiency editing and are therefore useful for distinguishing differences in on-target editing efficiency, combining this mutation with other amino acid changes identified in bacterial screens as double or triple mutants (i.e., combining 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) using Alt-R® crRNA:tracrRNA complexes targeting the HRPT 38509 locus (SEQ ID NO: 92). RNP complexes (10 nM) were delivered into HEK293 cells by reverse transfection using RNAiMAX, and DNA was extracted 48 hours later. Error bars represent standard error of the mean. [Figure 11]FIG. 1 shows exemplary results of experiments demonstrating that while the R691A mutant maintains high on-target editing activity at challenging on-target sites, combining this mutation with other amino acid changes identified in bacterial screens as double or triple mutants (i.e., combining 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) using Alt-R® crRNA:tracrRNA gRNA complexes (SEQ ID NO: 114) targeting the HEKSite4 locus. RNP complexes (10 nM) were delivered into HEK293 cells by reverse transfection using RNAiMAX, and DNA was extracted 48 hours later. Error bars represent standard error of the mean. On-target and off-target sites for the HEKSite4 locus are as described in FIG. 3. The experimental details and error analysis are similar to those described in FIG. [Figure 12]Figure 1 shows exemplary experimental results showing the effect of introducing all possible amino acid substitution mutations at the R691 position on editing at the HPRT 38509 site, with many substitutions maintaining on-target editing activity and others compromising editing efficiency. Cas9 protein was delivered by plasmid and CRISPR / Cas9 editing experiments used plasmid-mediated Cas9 expressing WT "R691" Cas9 (SEQ ID NO:5) or the indicated mutant Cas9s (SEQ ID NOs:7, 71-88) co-transfected with Alt-R® crRNA:tracrRNA complexes targeting the HPRT38509 locus (SEQ ID NO:92). Experimental conditions included 30 nM HPRT 38509 crRNA and 100 ng Cas9 plasmid introduced by lipofection with 0.5 microliters of Transit X-2 per well and cells were incubated for 48 hours prior to analysis. Error bars represent standard error of the mean. [Figure 13] Figure 1 shows exemplary experimental results showing the effect of introduction of all possible amino acid substitution mutations at the R691 position on editing at the HEKSite4 site, where the majority of the mutations dramatically reduce off-target editing while also showing retention of on-target activity. Cas9 protein was delivered by a plasmid and CRISPR / Cas9 editing experiments used plasmid-mediated Cas9 (expressing WT "R691" Cas9 (SEQ ID NO:5) or the indicated mutant Cas9s (SEQ ID NOs:7, 71-88)) co-transfected with Alt-R® crRNA:tracrRNA complexes targeting the HEKSite4 locus (SEQ ID NO:114). Experimental conditions included 30 nM HEKSite4 crRNA and 100 ng Cas9 plasmid introduced by lipofection with 0.5 microliters of Transit X-2 per well, and cells were incubated for 48 hours prior to analysis. Error bars represent standard error of the mean. On-target and off-target sites for the HEKSite4 locus are as described in Figure 3. The experimental details and error analysis are similar to those described in FIG. [Figure 14]Figure 1 shows exemplary results of an experiment demonstrating that selected Cas9 mutant proteins with different amino acid substitutions at the R691 position maintain on-target editing activity at different guide sites in the human HPRT gene. RNP complexes were formed (1 μM) with WT (R691), R691A, R691D, R691G, R691H, R691Y or R691W Cas9 proteins (SEQ ID NOs: 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 (SEQ ID NOs: 92 and 94). RNP complexes (10 nM) were delivered into HEK293 cells by reverse transfection using RNAiMAX, and DNA was extracted 48 hours later. Error bars represent standard error of the mean. Experimental details and error analysis are similar to those described in Figure 9. [Figure 15] Figure 1 shows exemplary results of an experiment 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 (1 μM) with WT (R691), R691A, R691D, R691G, R691H, R691Y or R691W Cas9 proteins (SEQ ID NOs: 5, 7, 73, 77, 78, 87 and 86, respectively) using Alt-R® crRNA:tracrRNA gRNA complexes targeting the HEKSite4 (SEQ ID NO: 114) or EMX1 (SEQ ID NO: 113) loci. RNP complexes (10 nM) were delivered into HEK293 cells by reverse transfection using RNAiMAX, and DNA was extracted 48 hours later. On-target and off-target sites for the EMX1 and HEKSite4 loci are described in Figures 2 and 3, respectively. Experimental details and error analysis are similar to those described in Figure 9. [Figure 16]Figure 1 shows an exemplary reduction of off-target editing activity in unbiased and genome-wide settings using a previously described GUIDE-Seq procedure (Tsai et al., Nature Biotechnology 33:187-197, 2015) in live cells. RNP complexes were formed with WT (SEQ ID NO:6) or R691A (SEQ ID NO:8) Cas9 proteins (1 μM) using Alt-R® crRNA:tracrRNA gRNA complexes targeting either EMX1 (SEQ ID NO:113), VEGFA3 (SEQ ID NO:116) or AR (SEQ ID NO:115) gene guide sites. 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 DNA was extracted 48 hours later. NGS library construction, sequencing and data analysis were performed as previously described (Tsai et al., Nature Biotechnology 33:187-197, 2015). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0023] The term "wild-type Cas9 protein" ("WT-Cas9" or "WT-Cas9 protein") encompasses a protein that has the identical amino acid sequence of a naturally occurring Streptococcus pyogenes Cas9 (e.g., SEQ ID NO:5) and has biochemical and biological activity when combined with an 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 comprising a wild-type Cas9 protein and an appropriate gRNA.

[0025] The phrase "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 comprising a mutant Cas9 protein that exhibits reduced off-target editing activity, typically greater than the reduced on-target editing activity, compared to the corresponding off-target and on-target editing activity of a wild-type CRISPR / Cas endonuclease system comprising a wild-type Cas9 protein, when both CRISPR / Cas endonuclease systems comprise the same gRNA for a given target sequence. The preferred off-target and on-target editing activities of the CRISPR / Cas endonuclease system depend on the gRNA and target sequence of interest, and such preferred off-target and on-target editing activities of the CRISPR / Cas endonuclease system with a mutant Cas9 protein are 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 that have biochemical and biological activity when combined with an appropriate guide RNA (e.g., sgRNA or dual crRNA:tracrRNA compositions) 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 in the sequence listing. However, for brevity, abbreviated mutant Cas9 protein amino acid code nomenclature is provided herein, and the position and identity of a given substitution mutation is provided relative to the position and identity of the amino acid in 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 replacing an arginine at residue 691 in the wild-type Cas9 protein amino acid sequence. For example, the specific single substitution mutation R691A refers to a mutant Cas9 protein amino acid sequence that includes an alanine instead of an arginine at residue 691 in the wild-type Cas9 protein amino acid sequence (see, e.g., SEQ ID NO: 7).

[0028] The mutated Cas9 proteins of the invention are active in 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 a wild-type CRISPR / Cas endonuclease system. As used herein, "mutated Cas9 proteins" excludes the mutant Cas9 proteins disclosed in Slaymaker et al., Science, 2016, 351, 84-88; Kleinstiver et al., Nature, 2016, 529, 490-495; Chen et al., Nature, 2017, http: / / dx.doi.org / 10.1038 / nature24268 (2017)), to the extent that these mutant Cas9 proteins, particularly when included in a CRISPR / Cas endonuclease system, exhibit reduced off-target editing activity and maintained on-target editing activity compared to a wild-type CRISPR / Cas endonuclease system, 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 provisos, a "mutated Cas9 protein" refers to, among others, 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 The present invention excludes mutant Cas9 proteins selected from the group consisting of SpCas9-HF1 (N497A / R661A / Q695A / Q926A), 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).

[0029] The term "polypeptide" refers to any linear or branched peptide containing more than one amino acid. Polypeptide includes proteins or fragments thereof 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 not native to a protein, which is covalently attached to the protein. Such additional amino acid information may include a tag that allows purification or identification of the fusion protein. Such additional amino acid information may include a peptide that allows 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 allows for biotinylation by the enzyme BirA so that the protein can be isolated with streptavidin; Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL), a peptide to which the protein calmodulin is attached; Polyglutamate tag (EEEEEE), a peptide that binds efficiently to anion exchange resins such as Mono-Q; E tag (GAPVPYPDPLEPR), a peptide that is recognized by an antibody; FLAG tag (DYKDDDDK), a peptide that is recognized by an antibody; HA tag (YPYDVPDYA), a peptide derived from hemagglutinin that is recognized by an antibody; the His tag (HHHHHH); the Myc tag (EQKLISEEDL), a peptide derived from c-myc recognized by an antibody; the 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 affinity purification of recombinant proteins; the S tag (KETAAAKFERQHMDS), a peptide derived from ribonuclease A; the SBP tag, a peptide that binds to streptavidin; (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP); Softag1 (SLAELLNAGLGGS), intended for mammalian expression; and Softag3 (TQDPSRVG), intended for prokaryotic expression;Strep tag (Strep tag II: WSHPQFEK), a peptide that binds to streptavidin or modified streptavidin called streptactin; TC tag (GKPIPNPLLGLDST), a tetracysteine ​​tag that is recognized by FlAsH and ReAsH biarsenical compounds (CCPGCC) V5 tag, a peptide that is recognized by antibodies; VSV tag (YTDIEMNRLGK), a peptide that is recognized by antibodies; Xpress tag (DLYDDDDK); Isopeptag (TDKDMTITFTNKKDAE), a peptide that is covalently attached to the pyrin-C protein; SpyTag (AHIVMVDA YKPTK; SnoopTag (KLGDIEFIKVNK), a peptide covalently attached to the SnoopCatcher protein; BCCP (biotin carboxyl carrier protein), a protein domain that is biotinylated by BirA to enable recognition by streptavidin; glutathione-S-transferase tag, a protein that is bound to immobilized glutathione; green fluorescent protein tag, a protein that is naturally fluorescent and can be bound by antibodies; HaloTag, a mutant bacterial haloalkane dehalogenase that is covalently attached to a reactive haloalkane substrate to enable attachment to a wide variety of substrates; maltose binding protein tag, a protein that is bound to amylose agarose; Nus tag; thioredoxin tag;and Fc tags derived from immunoglobulin Fc domains that allow dimerization and solubilization and can be used for purification on protein A sepharose. Nuclear localization signals (NLS), such as those obtained from SV40, allow proteins to be transported to the nucleus upon entry into the cell. Given 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 recombinant Cas9 proteins is expected to show improved genome editing activity when used in eukaryotic cells where the target genomic DNA substrate is present in the nucleus. Those skilled in the art understand how to make and use these various fusion tag technologies as well as fusion proteins that contain them.;

[0031] The term "isolated nucleic acid" includes DNA, RNA, cDNA, and vectors encoding them, free of other biological material from which they may be derived or associated, such as cellular components. Typically, isolated nucleic acids are purified from other biological material from which they may be derived or associated, such as cellular components.

[0032] The term "isolated wild-type Cas9 nucleic acid" refers to an isolated nucleic acid that encodes a 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," as the term modifies RNA, refers to a shortened or truncated form of the reference RNA that lacks a nucleotide sequence or an extended form of the reference RNA that includes additional nucleotide sequences.

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

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

[0037] Applicant has previously presented novel crRNA and tracrRNA oligonucleotide compositions that exhibit potent activity in the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) endonuclease system. The oligonucleotide compositions include length-modified forms of crRNA and tracrRNA as well as chemically modified forms of crRNA and tracrRNA. The length-modified forms of crRNA and tracrRNA allow for the preparation of active forms of these RNAs using routinely available cost-effective and efficient oligonucleotide synthesis protocols. The chemically modified forms of crRNA and tracrRNA provide active agents that can be tailored to certain unique properties, such as improved stability in cellular and in vivo environments or reduced risk of eliciting an innate immune response in mammalian cells. The length-modified forms of crRNA and tracrRNA may also contain modifications, thereby allowing access to a wide range of compositions that are active in the environment of the CRISPR-Cas endonuclease system. These oligonucleotide compositions and their properties in the CRISPR-Cas endonuclease system can be used 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 Serial No. 14 / 975,709, filed December 18, 2015, entitled "CRISPR-BASED COMPOSITIONS AND METHODS OF USE," to Collingwood et al. (Applicant: Integrated DNA Technologies, Inc., Skokie, IL (US)), published June 23, 2016 as U.S. Patent Publication No. US2016-0177304 A1, now issued as U.S. Patent No. __________, the contents of which are incorporated herein by reference in their entirety.

[0038] Mutant Cas9 proteins with reduced off-target gene editing activity 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 ("CRISPR / Cas endonuclease system"). The resulting CRISPR / Cas endonuclease system exhibits reduced off-target editing activity and maintained on-target editing activity compared to wild-type CRISPR / Cas endonuclease system.

[0039] Preferred single mutant Cas9 proteins comprise a substitution 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, 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 can 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 a wild-type CRISPR / Cas endonuclease system.

[0040] A preferred double mutant Cas9 protein comprises 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. Highly preferred doubly mutant Cas9 proteins comprise 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. Exemplary double mutant Cas9 proteins comprise two different 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. Exemplary double mutant Cas9 proteins include at least one member selected from the group consisting of SEQ ID NOs: 39-88. Additional substitution mutations can 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 the resulting 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.

[0041] In a second aspect, an isolated ribonucleoprotein complex is provided. The RNP comprises a mutant Cas9 protein and a gRNA complex. In a first respect, the gRNA comprises a stoichiometric (1:1) ratio of crRNA and tracrRNA. In a second respect, 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 respect, the gRNA comprises an sgRNA. Preferred mutant Cas9 proteins include those described above.

[0042] In a third aspect, an isolated nucleic acid encoding a mutant Cas9 protein is provided. A preferred isolated nucleic acid encodes the mutant Cas9 protein described above. An exemplary isolated nucleic acid encoding a mutant Cas9 protein can be easily generated from a nucleic acid encoding a wild-type Cas9 protein using recombinant DNA procedures or chemical synthesis methods. Preferred nucleic acids for this purpose include nucleic acids optimized for 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 NO: 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 NO: 3 and 4, respectively. Additionally, the present invention contemplates fusion proteins of WT-Cas9 and mutant Cas9, in which the coding sequences of WT-Cas9 and mutant Cas9 are fused to an amino acid sequence that encodes a nuclear localization ("NLS") of the fusion protein in eukaryotic cells or an amino acid sequence that facilitates purification of the protein. Exemplary fusion proteins comprising either the WT-Cas9 amino acid sequence or a mutant Cas9 amino acid sequence (e.g., the R691A mutant Cas9 protein) include SEQ ID NOs:6 and 8, respectively.

[0043] In the first aspect, the isolated nucleic acid comprises an mRNA encoding one of the aforementioned mutant Cas9 proteins. In the second aspect, the isolated nucleic acid comprises a DNA encoding a gene for one of the aforementioned mutant Cas9 proteins. A preferred DNA comprises 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 an appropriate expression vector to produce a cell line that constitutively or inducibly expresses the mutant Cas9. The aforementioned method can also be applied to embryos to produce offspring animals that constitutively or inducibly express the mutant Cas9.

[0044] In a fourth aspect, 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 a bacterial expression vector and a eukaryotic expression vector. In a third aspect, the CRISPR / Cas endonuclease system comprises a ribonucleoprotein complex comprising a mutant Cas9 protein and a gRNA complex. In a first aspect, the gRNA comprises a stoichiometric (1:1) ratio of crRNA and tracrRNA. In a second respect, the crRNA comprises an Alt-R® crRNA (Integrated DNA Technologies, Inc. (Skokie, IL (US)) directed to a specific editing target site for a given locus, and the tracrRNA comprises an Alt-R® tracrRNA (Integrated DNA Technologies, Inc. (Skokie, IL (US)). In another respect, the gRNA comprises an sgRNA.

[0045] In a fifth aspect, a method for performing gene editing is provided that reduces off-target editing activity and / or increases on-target editing activity. The method comprises contacting a candidate editing target site locus with an active CRISPR / Cas endonuclease system having a mutant Cas9 protein. In a first aspect, the 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 can 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 a wild-type CRISPR / Cas endonuclease system.

[0046] In another aspect, the method includes 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. Highly preferred doubly mutant Cas9 proteins comprise 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. Exemplary double mutant Cas9 proteins comprise two different 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. Exemplary double mutant Cas9 proteins include at least one member selected from the group consisting of SEQ ID NOs: 39-88. Additional substitution mutations can 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 the resulting 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.

[0047] The applications of Cas9-based tools are many and varied and include, but are not limited to, plant gene editing, yeast gene editing, mammalian gene editing, cell editing in living animal organs, embryo editing, rapid generation of knock-out / 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 code / express the same amino acid (AA) sequence, since in many cases more than one codon can code for the same amino acid. The DNA sequences shown below are given as examples only, and other DNA sequences that code for the same protein (e.g., the same amino acid sequence) are contemplated. It is further understood that additional features, elements, or tags, such as NLS domains, may be added to the sequence. 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- and N-terminal SV40NLS domains and HIS tags. For other Cas9 variants, only amino acid sequences are provided, but it is contemplated that similar additions of NLS and His tag domains may be added to facilitate use in producing recombinant proteins for use in mammalian cells. Mutations that differ from the WT sequence are clearly indicated using bold underlined font.

[0049] SEQ ID NO:1 WT SpyCas9 DNA sequence codon-optimized for expression in E. coli.

[0050] [ka] TIFF2024178257000003.tif147154

[0051] SEQ ID NO:2 WT SpyCas9 DNA sequence codon-optimized for expression in H. sapiens.

[0052] [ka] TIFF2024178257000005.tif175158

[0053] SEQ ID NO:3 Codon-optimized R691A mutant SpyCas9 DNA sequence for expression in E. coli.

[0054] [ka] TIFF2024178257000007.tif241155

[0055] SEQ ID NO:4 Codon-optimized R691A mutant SpyCas9 DNA sequence for expression in H. sapiens.

[0056] [ka] TIFF2024178257000009.tif26156

[0057] SEQ ID NO:5 WT SpyCas9 AA sequence.

[0058] [ka]

[0059] SEQ ID NO:6 WT SpyCas9 AA sequence with the NLS domain and HIS-Tag purification domain appended.

[0060] [ka]

[0061] SEQ ID NO:7 R691A mutated SpyCas9 AA sequence.

[0062] [ka]

[0063] SEQ ID NO:8 R691A mutant SpyCas9 AA sequence with the NLS domain and HIS-Tag purification domain added.

[0064] [ka]

[0065] SEQ ID NO:9 R494C mutant SpyCas9 AA sequence.

[0066] [ka]

[0067] SEQ ID NO:10 R494A mutated SpyCas9 AA sequence.

[0068] [ka]

[0069] SEQ ID NO:11 N522K mutated SpyCas9 AA sequence.

[0070] [ka]

[0071] SEQ ID NO:12 N522A mutated SpyCas9 AA sequence.

[0072] [ka]

[0073] SEQ ID NO:13 N588D mutant SpyCas9 AA sequence.

[0074] [ka]

[0075] SEQ ID NO:14 N588A mutated SpyCas9 AA sequence.

[0076] [ka]

[0077] SEQ ID NO:15 N612A mutated SpyCas9 AA sequence.

[0078] [ka]

[0079] SEQ ID NO:16 T657A mutant SpyCas9 AA sequence.

[0080] [ka]

[0081] SEQ ID NO:17 S663A mutated SpyCas9 AA sequence.

[0082] [ka]

[0083] SEQ ID NO:18 N692D mutated SpyCas9 AA sequence.

[0084] [ka]

[0085] SEQ ID NO:19 N692A mutated SpyCas9 AA sequence.

[0086] [ka]

[0087] SEQ ID NO:20 S730G mutant SpyCas9 AA sequence.

[0088] [ka]

[0089] SEQ ID NO:21 S730A mutant SpyCas9 AA sequence.

[0090] [ka]

[0091] SEQ ID NO:22 T740A mutant SpyCas9 AA sequence.

[0092] [ka]

[0093] SEQ ID NO:23 R765G mutant SpyCas9 AA sequence.

[0094] [ka]

[0095] SEQ ID NO:24 R765A mutated SpyCas9 AA sequence.

[0096] [ka]

[0097] SEQ ID NO:25 T770K mutant SpyCas9 AA sequence.

[0098] [ka] SEQ ID NO:26 T770A mutant SpyCas9 AA sequence.

[0099] [ka]

[0100] SEQ ID NO:27 N776A mutated SpyCas9 AA sequence.

[0101] [ka]

[0102] SEQ ID NO:28 R778A mutated SpyCas9 AA sequence.

[0103] [ka]

[0104] SEQ ID NO:29 R783A mutated SpyCas9 AA sequence.

[0105] [ka]

[0106] SEQ ID NO:30 S793A mutated SpyCas9 AA sequence.

[0107] [ka]

[0108] SEQ ID NO:31 N803D mutated SpyCas9 AA sequence.

[0109] [ka]

[0110] SEQ ID NO:32 N803A mutated SpyCas9 AA sequence.

[0111] [ka]

[0112] SEQ ID NO:33 S845A mutated SpyCas9 AA sequence.

[0113] [ka]

[0114] SEQ ID NO:34 N854K mutant SpyCas9 AA sequence.

[0115] [ka]

[0116] SEQ ID NO:35 N854A mutated SpyCas9 AA sequence.

[0117] [ka]

[0118] SEQ ID NO:36 S872A mutated SpyCas9 AA sequence.

[0119] [ka]

[0120] SEQ ID NO:37 R925C mutant SpyCas9 AA sequence.

[0121] [ka]

[0122] SEQ ID NO:38 R925A mutated SpyCas9 AA sequence.

[0123] [ka]

[0124] SEQ ID NO:39 R691A / N692A mutated SpyCas9 AA sequence.

[0125] [ka]

[0126] SEQ ID NO:40 R691A / R494C mutated SpyCas9 AA sequence.

[0127] [ka]

[0128] SEQ ID NO:41 N692A / R494C mutated SpyCas9 AA sequence.

[0129] [ka]

[0130] SEQ ID NO:42 R691A / N522K mutant SpyCas9 AA sequence.

[0131] [ka]

[0132] SEQ ID NO:43 N692A / N522K mutant SpyCas9 AA sequence.

[0133] [ka]

[0134] SEQ ID NO:44 R691A / N588D mutant SpyCas9 AA sequence.

[0135] [ka]

[0136] SEQ ID NO:45 N692A / N588D mutant SpyCas9 AA sequence.

[0137] [ka]

[0138] SEQ ID NO:46 R691A / N612A mutated SpyCas9 AA sequence.

[0139] [ka]

[0140] SEQ ID NO:47 N692A / N612A mutant SpyCas9 AA sequence.

[0141] [ka]

[0142] SEQ ID NO:48 R691A / S663A mutated SpyCas9 AA sequence.

[0143] [ka]

[0144] SEQ ID NO:49 N692A / S663A mutated SpyCas9 AA sequence.

[0145] [ka]

[0146] SEQ ID NO:50 R691A / S730G mutant SpyCas9 AA sequence.

[0147] [ka]

[0148] SEQ ID NO:51 N692A / S730G mutant SpyCas9 AA sequence.

[0149] [ka]

[0150] SEQ ID NO:52 R691A / T740A mutant SpyCas9 AA sequence.

[0151] [ka]

[0152] SEQ ID NO:53 N692A / T740A mutant SpyCas9 AA sequence.

[0153] [ka]

[0154] SEQ ID NO:54 R691A / R765G mutant SpyCas9 AA sequence.

[0155] [ka]

[0156] SEQ ID NO:55 N692A / R765G mutant SpyCas9 AA sequence.

[0157] [ka]

[0158] SEQ ID NO:56 R691A / T770K mutant SpyCas9 AA sequence.

[0159] [ka]

[0160] SEQ ID NO:57 N962A / T770K mutant SpyCas9 AA sequence.

[0161] [ka]

[0162] SEQ ID NO:58 R691A / S793A mutated SpyCas9 AA sequence.

[0163] [ka]

[0164] SEQ ID NO:59 N692A / S793A mutated SpyCas9 AA sequence.

[0165] [ka]

[0166] SEQ ID NO:60 R691A / N803D mutant SpyCas9 AA sequence.

[0167] [ka]

[0168] SEQ ID NO:61 N692A / N803D mutated SpyCas9 AA sequence.

[0169] [ka]

[0170] SEQ ID NO:62 R691A / S845A mutant SpyCas9 AA sequence.

[0171] [ka]

[0172] SEQ ID NO:63 N692A / S845A mutant SpyCas9 AA sequence.

[0173] [ka]

[0174] SEQ ID NO:64 R691A / N854K mutant SpyCas9 AA sequence.

[0175] [ka]

[0176] SEQ ID NO:65 N692A / N854K mutant SpyCas9 AA sequence.

[0177] [ka]

[0178] SEQ ID NO:66 R691A / S872A mutant SpyCas9 AA sequence.

[0179] [ka]

[0180] SEQ ID NO:67 N692A / S872A mutant SpyCas9 AA sequence.

[0181] [ka]

[0182] SEQ ID NO:68 R691A / N692A / T740A mutant SpyCas9 AA sequence.

[0183] [ka]

[0184] SEQ ID NO:69 R691A / N692A / S845A mutant SpyCas9 AA sequence.

[0185] [ka]

[0186] SEQ ID NO:70 R691A / N692A / S872A mutant SpyCas9 AA sequence.

[0187] [ka]

[0188] SEQ ID NO:71 R691S mutated SpyCas9 AA sequence.

[0189] [ka]

[0190] SEQ ID NO:72 R691N mutated SpyCas9 AA sequence.

[0191] [ka]

[0192] SEQ ID NO:73 R691D mutated SpyCas9 AA sequence.

[0193] [ka]

[0194] SEQ ID NO:74 R691C mutant SpyCas9 AA sequence.

[0195] [ka]

[0196] SEQ ID NO:75 R691Q mutated SpyCas9 AA sequence.

[0197] [ka]

[0198] SEQ ID NO:76 R691E mutated SpyCas9 AA sequence.

[0199] [ka]

[0200] SEQ ID NO:77 R691G mutated SpyCas9 AA sequence.

[0201] [ka]

[0202] SEQ ID NO:78 R691H mutant SpyCas9 AA sequence.

[0203] [ka]

[0204] SEQ ID NO:79 R691I mutated SpyCas9 AA sequence.

[0205] [ka]

[0206] SEQ ID NO:80 R691L mutated SpyCas9 AA sequence.

[0207] [ka]

[0208] SEQ ID NO:81 R691K mutant SpyCas9 AA sequence.

[0209] [ka]

[0210] SEQ ID NO:82 R691M mutated SpyCas9 AA sequence.

[0211] [ka]

[0212] SEQ ID NO:83 R691F mutated SpyCas9 AA sequence.

[0213] [ka]

[0214] SEQ ID NO:84 R691P mutated SpyCas9 AA sequence.

[0215] [ka]

[0216] SEQ ID NO:85 R691T mutant SpyCas9 AA sequence.

[0217] [ka]

[0218] SEQ ID NO:86 R691W mutant SpyCas9 AA sequence.

[0219] [ka]

[0220] SEQ ID NO:87 R691Y mutated SpyCas9 AA sequence.

[0221] [ka]

[0222] SEQ ID NO:88 R691V mutated SpyCas9 AA sequence.

[0223] [ka]

[0224] [Example 2] Bacterial genetic screen to enrich for mutant Cas9 peptides that retain high on-target activity while reducing off-target cleavage. The following example details a genetic screen performed in E. coli to identify candidate Cas9 mutant enzymes of interest from a library of approximately 250,000 mutant clones for subsequent more detailed characterization.

[0225] All of the Cas9 mutants with reduced off-target effects identified in the prior art were developed using a rational design approach by making alanine substitutions of charged amino acids close to the Cas9 nucleic acid binding pocket based on previously published crystal and co-crystal structures of Cas9, Cas9-gRNA and Cas9-gRNA-DNA complexes. This limits the sequence space available for mutations to a very limited number of amino acid residues. The present invention instead identifies novel useful Cas9 mutants using unbiased screening of a large number of random mutants generated by low-fidelity PCR of Cas9 expression cassettes, which greatly expands the potential sequence space explored for useful mutations.

[0226] In the present invention, we used a bacterial screening approach to select any amino acid substitution in Cas9 that would facilitate on-target cleavage but avoid off-target cleavage. The screening approach was adapted from screens previously performed for other applications. See Chen and Zhao (Nucleic Acids Research, 33(18)pe154, 2005) and Kleinstiver et al. (Nature, 523, 481-485, 2015). The screen was based on co-transformation of E. coli cells with 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 randomly mutagenized (approximately 6 mutations per kilobase) cas9 sequences, a single guide RNA (sgRNA) specific for each on-target site, and known off-target cleavage sites for each guide RNA (SEQ ID NOs: 134, 136, 138) (coupled with chloramphenicol expression such that if cleavage occurs, the resistance gene is not expressed and the cells die when exposed to the selectable marker, chloramphenicol). The screen design allows for the sequential use of different on-target sites (toxins) paired with appropriate off-target sites (chloramphenicol), so that the screen can be performed iteratively to ensure that isolates were not selected solely based on performance against a single gRNA target site.

[0227] The screening procedure 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 is produced and cell survival is possible. Cells carrying the stably replicating toxin plasmid were then transformed with a chloramphenicol Cas9-sgRNA off-target plasmid, grown non-selectively at 37°C for 1 hour, recovered, and then transformed bacteria were plated on selective media containing both chloramphenicol and arabinose. The bacteria grown were i) those successfully transformed with the Cas9-sgRNA off-target plasmid, ii) those that expressed enough Cas9 and VEGFA3 sgRNA to cleave the toxin on-target plasmid, and iii) those that avoided cleavage of the chloramphenicol Cas9-sgRNA off-target plasmid enough to allow sufficient chloramphenicol resistance to survive under selection. A pool of candidate mutations that allowed survival with all three tested guides (VEGFA3, HEKSite4, and EMX1) was generated. Within this pool, mutations that were isolated multiple times throughout the screening process (a total of 94 clones) were carried forward for further analysis in mammalian cells. A schematic of the screening method is shown in FIG. 1.

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

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

[0230] Alt-R® SpCas9 expression plasmid (WT) was modified by site-directed mutagenesis and confirmed to carry the indicated mutations by DNA sequencing. CRISPR / Cas9 experiments were performed using two copies of Alt-R® crRNA and tracrRNA systems co-transfected simultaneously with different mutant WT and mutant Alt-R® Cas9 expression plasmids. Alt-R™ crRNA targeting NGG PAM-containing sequences in EMX1 and HEKSite4 loci (Table 1, SEQ ID NOs: 113 and 114) was duplexed to Alt-R® tracrRNA at a 1:1 molar ratio (3 μM) by heating to 95° C. for 5 min followed by slow cooling to 25° C. Reverse transfections were performed in triplicate with 0.5 μl of Transit-X2 (Mirus Bio LLC), 30 nM of EMX1 or HEKSite4 Alt-R® gRNA complexes and 0.1 μg of Alt-R® Cas9 plasmid (containing WT or the indicated mutants). Transfection lipid complexes were formed for 20 min at room temperature according to the manufacturer's instructions, and 40,000 HEK293 cells were added to each transfection. After 48 h of incubation at 37° C. and 5% CO2, the attached 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 min, followed by heat inactivation at 98° C. for 3 min. The crude DNA samples were then diluted 3-fold with 0.1 ml of ddH2O and used as PCR templates. 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 fragments of ≦1 kb of either the EMX1 or HEKSite4 locus using the following cycle parameters: 95 cycles over 1 min, cooled to 85°C. 10:00 , cooled to 75°C in 1 minute. 1:00 , cooled to 65°C in 1 minute. 1:00 , cooled to 55°C in 1 minute65°C 1:00 , cooled to 45°C in 1 minute. 1:00 , cooled to 35°C for 1 minute. 1:00 , cooled to 25°C over 1 minute. 1:00 ,twenty five 1:00 Heteroduplexes were formed using X Y The aforementioned numbers, written as x, y, and y, represent a constant temperature (in degrees Fahrenheit) and a time period (minutes when expressed as "n:00" or seconds when expressed as "0:nn" (where n is an integer). Heteroduplexes were cleaved by adding 2U T7 endonuclease I (New England Biolabs) at 37°C for 1 hour, and cleavage products were analyzed by capillary electrophoresis (Fragment Analyzer, Advanced Analytical). A T7E1 mismatch cleavage assay was used to evaluate DNA editing efficiency in this and subsequent examples. The complete protocol has been described (see Jacobi et al., Methods, 121-122, pp. 16-28, 2017).

[0231] These results show 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 HEKSite4 loci (Figure 3). Many of the point mutations isolated from our screen resulted in substitutions to amino acids other than alanine. For those mutants from the initial screen that were non-alanine substitutions, we performed site-directed mutagenesis to change these mutations to alanine substitutions at these positions and compared these to both WT Cas9 and the original amino acid substitutions (new alanine mutants include SEQ ID NOs: 7, 10, 12, 14, 19, 21, 24, 26, 32, 35, and 38). We observed similar phenotypes between the original mutants isolated from the primary screen and the new alanine mutants (Figures 4 and 5). In some cases, the alanine substitutions resulted in higher on-target editing activity and lower off-target editing activity. In general, the alanine mutants performed similarly or better than the original mutants isolated, therefore combinations of forward mutations were tested only using alanine substitutions. Mutations at positions R691 and N692 showed the greatest reduction in off-target gene editing activity for both EMX1 and HEKSite4 crRNAs. Mutations at R691 demonstrate WT Cas9 levels of on-target editing activity, while mutations at N692 show a slight reduction in on-target editing activity for both crRNAs. For this reason, we chose to generate double and triple mutant combinations based on either the R691A or N692A mutation as a starting point. We also generated and tested a R691A / N692A double mutant. The new double mutant Cas9 sequences include SEQ ID NOs: 39 to 70. All combinations of these mutations had an additive effect in that no detectable off-target editing activity was detected (Figure 6).Two published prior art high fidelity Cas9 proteins, eSpCas9(1.1) (K848A, K1003A and R1060A) (Slaymaker et al., Science, 351, 84-88, 2016) and SpCas9-HF1 (N497A, R661A, Q695A and Q926A) (Kleinstiver et al., Nature, 490-495, 2016), also had undetectable off-target editing activity when delivered by plasmid, but reduced on-target editing activity compared to the wild-type protein. The mutants of the present invention have a superior overall editing activity profile when delivered as a plasmid compared to these mutants from the prior art. The sequences of the target-specific protospacer domains of all CRISPR gRNAs utilized in the examples are provided in Table 1. These RNA sequences represent the variable domains of the gRNA that vary with the target site. In practice, the protospacer domain is flanked by additional universal RNA sequences to comprise a complete functional Cas9 crRNA or sgRNA (see Jinek et al., Science, 2012, 337(6096):816-21 and Jacobi et al., Methods, 2017, 121-122, 16-28). The sequences of the DNA on-target and off-target domains and dsDNA GUIDE-Seq tags studied in the examples are shown in Table 2.

[0232] [Table 1] TIFF2024178257000095.tif240159TIFF2024178257000096.tif87158

[0233] [Table 2]

[0234] [Example 4] RNP delivery of the novel Cas9 mutant reduces off-target editing activity while maintaining on-target editing activity.

[0235] The following examples demonstrate the ability of the Cas9 variants of the 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 into the context of a Cas9 protein expression / purification plasmid that allows expression of recombinant proteins in E. coli, and the resulting protein contains an NLS domain to facilitate nuclear delivery in mammalian cells as well as a HIS tag to simplify purification (see WT Cas9 DNA sequence, SEQ ID NO:1 and R691A mutant, SEQ ID NO:3). The amino acid sequences with domain additions are shown as exemplary models (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 methods. The published high-fidelity Cas9 proteins, eSpCas9(1.1) and SpCas9-HF1, were also purified using this method. CRISPR / Cas9 experiments were performed by first forming 1 μM RNP complexes with purified Cas9 protein and two parts Alt-R®RNA (crRNA:tracrRNA complex) in Opti-MEM for 5 min at 25°C. Alt-R® crRNA targeted HPRT gene (SEQ ID NO: 89-100) and CTLA4 gene (SEQ ID NO: 101-112) and was delivered to HEK293 cells (40,000 cells / well) by reverse transfection of preformed RNP complexes using 1.2 μl of RNAiMAX. Experiments were performed in biological triplicates and cells were lysed after 48 h incubation at 37° C., 5% CO2. DNA extraction, PCR amplification and T7E1 digestion were performed as described for the plasmid-based experiments in Example 3. PCR amplification primers are listed in Table 1 (SEQ ID NO: 117-122).

[0237] Figure 7 shows genome editing efficiency using the RNP method in mammalian cells at 12 sites in the human HPRT gene comparing WT, mutant R691A, eSpCas9(1.1) and Cas9-HF1 proteins, and 12 sites in the human CTLA4 gene are shown in Figure 8. The R691A Cas9 mutant of the present invention showed on-target editing activity equivalent to wild-type Cas9 at 95% of the sites tested, 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 sites previously identified for crRNA sites in three different genes (EMX1 SEQ ID NO:113, HEKSite4 SEQ ID NO:114 and VEGFA3 SEQ ID NO:116). The on-target sites are shown aligned with the off-target sites in FIG. 9 (SEQ ID NOs:121-126, 129 and 130). Significant off-target editing activity was seen at all three sites for the WT Cas9 enzyme, while all three mutant Cas9 enzymes showed no detectable off-target activity using this assay. However, while the novel R691A mutant of the present invention showed identical on-target activity at these three sites, only the prior art mutants eSpCas9(1.1) and Cas9-HF1 showed full activity at the EMX1 site, with significantly reduced activity seen at the HEKSite4 and VEGF3A loci. This demonstrates the utility of the present invention: for the R691A Cas9 mutant, off-target activity is reduced while on-target activity is maintained at a high level, providing a significant improvement over the performance of existing mutants from the prior art.

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

[0240] Figure 10 shows that all of the Cas9 single mutants tested showed 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 same double mutants showed good activity when overexpressed from a plasmid template (Figure 6).

[0241] These mutants were also delivered as RNPs to test for off-target activity and the HEKSite4 locus. Overall, the N692A mutant performed similarly to R691A, only slightly reducing on-target editing activity (Figure 10) and undetectable off-target editing activity (Figure 11). Other single mutants in the isolates showed excellent on-target editing activity (Figure 10), but only slightly or no reduction in off-target editing activity at this difficult site (Figure 11). Multiple combinations of mutations containing R691A or N692A showed excellent on-target editing activity coupled with undetectable off-target editing activity using plasmid delivery, but these mutants showed reduced on-target editing activity using RNP delivery (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] Testing of additional amino acid mutations at position R691 using plasmid and RNP delivery methods. So far, site R691 has been characterized as WT and 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 position were introduced into a mammalian Cas9 expression plasmid using site-directed mutagenesis and tested for function in HEK293 cells using plasmid delivery (methods described in Example 3). On-target editing activity was studied using crRNA HPRT 38509 (SEQ ID NO: 92) and 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, whereas the WT and mutants utilizing the 14 other amino acids at this position all showed high activity.

[0244] The combination of on-target and off-target activity for this set of 20 Cas9 variants was studied in crRNA HEKSite4 (SEQ ID NO: 114), a highly active site for both on-target and off-target in WT Cas9. Figure 13 demonstrates that WT Cas9 and 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 to mutate to improve Cas9 function, and that a variety of different amino acid substitutions function well in this environment.

[0245] The best performing of these mutants, R691D, R691G, R691H, R691Y and R691W, were prepared as recombinant proteins and tested for on-target and off-target editing activity in comparison with 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 reduction in off-target activity for sites EMX1 and HEKSite4 (Figure 15), while low but detectable off-target editing activity was observed for the R691G, R691H and R691Y mutants. The R691A, R691D and R691W mutants provided the best combination of on-target editing activity and reduced off-target editing activity when both plasmid and RNP delivery methods were considered.

[0246] The R691A mutant was tested for global off-target effects using a publicly available unbiased genome-wide next-generation sequencing (NGS) assay called Guide-Seq (Tsai et al., Nature Biotechnology, 33, 187-197, 2015). This was the source for the validated off-target sites for EMX1, HEKSite4 and VEGF3A cRNAs studied in the above examples. The Guide-Seq protocol was performed as recommended using crRNA guides EMX1 (SEQ ID NO: 113), VEGFA3 (SEQ ID NO: 116) and AR (SEQ ID NO: 115) using either WT Cas9 or R691A mutant Cas9 with RNP delivery. NGS library construction and data processing were performed as previously described (Tsai et al., Nature Biotechnology 33:187-197, 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 in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0248] The use of the terms "a," "an," and "the," and similar referents in the context of describing the present invention (particularly in the context of the claims below) are to be construed as including both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated herein. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value contained within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise required. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0249] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors expect that those of skill in the art will employ such variations as appropriate, and the inventors intend for the invention to be carried out in ways other than those specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited within the scope of the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. An isolated mutant Cas9 protein that is active in a CRISPR / Cas endonuclease system, wherein the 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.

2. (a) a single substitution mutation introduced into a WT-Cas9 protein selected from 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.

2. The isolated mutant Cas9 protein of claim 1, comprising a substitution mutation selected from the group consisting of:

3. 2. The isolated mutant Cas9 protein of claim 1, comprising a substitution mutation selected from the group consisting of double substitution mutations introduced into a WT-Cas9 protein selected from two of 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. 2. The isolated mutant Cas9 protein of claim 1, selected from the group consisting of SEQ ID NOs: 7-38.

5. 2. The isolated mutant Cas9 protein of claim 1, selected from the group consisting of SEQ ID NOs: 39-88.

6. Mutant Cas9 proteins, and gRNA complex 1. 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 a wild-type CRISPR / Cas endonuclease system.

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

8. The gRNA is isolated crRNAs, including Alt-R™ crRNAs directed against specific editing target sites for a given locus; and tracrRNA, including Alt-R™ tracrRNA 7. The isolated ribonucleoprotein complex of claim 6, comprising:

9. The isolated ribonucleoprotein complex of claim 6, wherein the gRNA comprises an sgRNA.

10. The mutant Cas9 protein, (a) a single substitution mutation introduced into a WT-Cas9 protein selected from 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.

7. The isolated ribonucleoprotein complex of claim 6, comprising a substitution mutation selected from the group consisting of:

11. 7. The isolated ribonucleoprotein complex of claim 6, wherein the mutant Cas9 protein comprises a substitution mutation selected from the group consisting of a double substitution mutation 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.

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

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

14. An isolated nucleic acid encoding a mutant Cas9 protein, wherein the mutant Cas9 protein is active in a CRISPR / Cas endonuclease system, and wherein the 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.

15. The mutant Cas9 protein, (a) a single substitution mutation introduced into a WT-Cas9 protein selected from 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 a mutant Cas9 protein of claim 14, comprising a substitution mutation selected from the group consisting of a double substitution mutation introduced into the WT-Cas9 protein 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.

16. 15. An isolated nucleic acid encoding a mutant Cas9 protein of claim 14, wherein the mutant Cas9 protein comprises a substitution mutation selected from the group consisting of a double substitution mutation introduced into a 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.

17. 15. 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: 7-38.

18. 15. 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: 39-88.

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

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

21. 20. The CRISPR / Cas endonuclease system of claim 19, wherein the DNA expression vector comprises a plasmid-based vector.

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

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

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

25. 20. The CRISPR / Cas endonuclease system of claim 19, wherein the gRNA comprises an sgRNA.

26. The mutant Cas9 protein, (a) a single substitution mutation introduced into a WT-Cas9 protein selected from 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.

20. The CRISPR / Cas endonuclease system of claim 19, comprising a substitution mutation selected from the group consisting of:

27. The mutant Cas9 protein is 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; 20. The CRISPR / Cas endonuclease system of claim 19, comprising a substitution mutation selected from the group consisting of double substitution mutations introduced into the WT-Cas9 protein selected from two of positions: 783A; S793A; N803D or N803A; S845A; N854K or N854A; S872A; and R925C or R925A.

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

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

30. 1. A method for performing gene editing that reduces off-target editing activity and / or increases on-target editing activity, comprising: A method comprising contacting a candidate editing target site locus with an active CRISPR / Cas endonuclease system having a mutated 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. The mutant Cas9 protein, (a) a single substitution mutation introduced into a WT-Cas9 protein selected from 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.

31. The method of claim 30, comprising a substitution mutation selected from the group consisting of:

32. WT-Cas9 protein, wherein 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.

31. The method of claim 30, comprising a substitution mutation selected from the group consisting of a double substitution mutation introduced into

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

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

Images