Mutant CAS9 protein

Abstract

To provide methods of making deletion mutants of Cas9 proteins and methods of making chimeric Cas9 proteins.SOLUTION: To analyze activity of Cas9, a series of domain exchange mutants were made by replacing the N and / or C terminus of NM (Neisseria meningitides Cas9) with the homologous region from STI (Streptococcus thermophilus Cas9). Then the guide RNA and / or Cas9 specific protospacer-adjacent motif (PAM) within the reporter are altered to determine the influence of the domain exchanges of the chimeric proteins. The C-terminal exchange generated a NM-STI hybrid that was capable of interacting with the STI crRNAitracrRNA complex and was further able to suppress a reporter with a STI specific PAM.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 61 / 906,374, filed November 19, 2013, which is incorporated by reference in its entirety for all purposes.

[0002] Statement of Government Interest This invention was made with Government support under Grant No. P50HG005550 from the National Institutes of Health. The United States Government has certain rights in this invention. [Background technology]

[0003] Bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs complexed with Cas proteins to degrade complementary sequences present in invading foreign nucleic acids. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602-607 (2011); Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America 109, E2579-2586 (2012); Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012); Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR / Cas system provides immunity in Escherichia coli. Nucleic acids research 39, 9275-9282 (2011); and Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annual review of genetics 45, 273-297 (2011).Recently, in vitro reconstitution of the type II CRISPR system in S. pyogenes demonstrated that a crRNA ("CRISPR RNA") fused to a tracrRNA ("trans-activating CRISPR RNA"), normally encoded in trans, is sufficient to direct the Cas9 protein to sequence-specifically cleave a target DNA sequence that matches the crRNA. Expression of a gRNA homologous to the target site recruits Cas9 and degrades the target DNA. See H. Deveau et al, Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology 190, 1390 (Feb, 2008). Summary of the Invention [Problem to be solved by the invention]

[0004] Cas9 is a DNA nuclease that can be programmed to target almost any region of the genome by expressing a guide RNA (gRNA) that contains 20 base pairs complementary to the genomic region desired to be targeted and a motif that recruits Cas9. All characterized and putative Cas9 family members are several kilobases (>3000 base pairs) in size, with the smallest functionally validated member, NM-Cas9 (Cas9 from Neisseria meningitides), being 3,249 base pairs in size. The large size of this protein makes it difficult to deliver and manipulate, limiting its potential for biotechnological and therapeutic applications. [Means for solving the problem]

[0005] An embodiment of the present disclosure relates to an RNA-guided DNA-binding protein of a type II CRISPR system that binds to DNA and is guided by one or more RNAs, which is modified to omit a portion of the protein, but functions as an RNA-guided DNA-binding nuclease that can bind to target DNA and form double-stranded breaks in the target DNA. According to one embodiment, the RNA-guided DNA-binding protein of the type II CRISPR system is a Cas9 protein.

[0006] An embodiment of the present disclosure relates to an RNA-guided DNA-binding protein of the type II CRISPR system that is modified to delete a portion of the protein but functions as an RNA-guided DNA-binding nickase that can bind to target DNA and form a single-stranded break or nick in the target DNA. According to an embodiment, the RNA-guided DNA-binding protein of the type II CRISPR system is a Cas9 protein.

[0007] The embodiment of the present disclosure relates to the RNA-guided DNA-binding protein of type II CRISPR system, which is modified to delete a part of the protein, but is nuclease null, i.e., the RNA-guided DNA-binding protein lacks nuclease activity, and functions as the RNA-guided DNA-binding protein.According to one embodiment, the RNA-guided DNA-binding protein of type II CRISPR system is Cas9 protein.

[0008] According to an embodiment, portions of RNA-guided DNA-binding proteins are identified for deletion by identifying RNA-guided DNA-binding protein sequences that are less conserved or highly diverged within a particular RNA-guided DNA-binding protein family and / or protein sequences between the boundary between low and high conservation, referred to herein as "conservation edge", within a particular RNA-guided DNA-binding protein family, within a population of species. According to this embodiment, amino acid sequences within a DNA-binding protein, such as an RNA-guided DNA-binding protein, such as Cas9, are identified as being highly conserved or low conserved using methods as described herein and known to those skilled in the art. According to an embodiment, the highly conserved amino acid sequence and the low conserved amino acid sequence are adjacent, e.g., directly adjacent, to each other in the protein sequence of the DNA-binding protein as a whole. The highly conserved amino acid sequence and the low conserved amino acid sequence are distinguished by an amino acid that separates the highly conserved amino acid sequence from the low conserved amino acid sequence. Thus, an amino acid that separates a highly conserved amino acid sequence from a less conserved amino acid sequence is referred to herein as an "edge amino acid" or "conserved edge" so long as it is an edge or terminal portion of either the highly conserved or less conserved amino acid sequence. Thus, the disclosed method contemplates the identification of an amino acid that separates or distinguishes a highly conserved amino acid sequence from a less conserved amino acid sequence. Such an amino acid is referred to herein as an "edge amino acid." According to this aspect, a pair of edge amino acids may be adjacent or bordering on either end of a highly conserved amino acid sequence. Similarly, a pair of edge amino acids may be adjacent or bordering on either end of a less conserved amino acid sequence. Further according to this aspect, certain exemplary embodiments relate to the identification of highly conserved adjacent amino acid sequences and the identification of less conserved amino acid sequences within the protein sequence of an entire DNA binding protein.In particular, certain exemplary embodiments relate to the identification of highly conserved sequences in tandem or tandem with less conserved sequences within the protein sequence of the entire DNA binding protein, in particular the identification of highly conserved (HC) sequences flanked at either end by less conserved (LC) sequences or less conserved (LC) sequences flanked at either end by highly conserved (HC) sequences. Thus, exemplary tandem or tandem sequences identified by the methods described herein may be represented diagrammatically as LC-HC-LC or HC-LC-HC. According to this aspect, a highly or less conserved central sequence is flanked by less or more conserved flanking sequences, respectively. In the exemplary tandem sequence LC-HC-LC, a pair of edge amino acids separates or separates a highly conserved (HC) amino acid sequence from two adjacent less conserved (LC) amino acid sequences that are either at either end of or adjacent to the highly conserved amino acid sequence. In an exemplary tandem sequence HC-LC-HC, a pair of edge amino acids separates or separates a less conserved (LC) amino acid sequence from two adjacent highly conserved (HC) amino acid sequences that are either on either side of the less conserved amino acid sequence or adjacent to the less conserved amino acid sequence. When such an exemplary tandem sequence is identified using the methods described herein, whether the central sequence is a highly conserved or a less conserved amino acid sequence, the central sequence is removed according to the methods described herein to create a mutant DNA-binding protein that maintains DNA-binding activity and is smaller in size than the wild-type DNA-binding protein. The edge amino acids define the central sequence that is removed to create the mutant DNA-binding protein by flanking the central sequence or by separating the central sequence from the adjacent sequences in tandem. According to certain aspects of the present disclosure, the central sequence in the tandem sequence can be removed regardless of whether the central sequence is a highly conserved or a less conserved amino acid sequence, so long as the mutant DNA-binding protein retains useful DNA-binding protein activity.

[0009] To identify the less conserved sequences or protein sequences between the conservation edges in this disclosure, alignment is made from a collection of sequences obtained from PFAM or from database searches of Cas9 homologues. This alignment is adjusted computationally, and the degree of conservation is calculated as the (relative) entropy of amino acid frequency per position. Next, the sequence is removed from the RNA-guided DNA binding protein to generate mutants.

[0010] In one embodiment, to identify regions within the multi-domain Cas9 protein that may be suitable for deletion, a bioinformatics approach is used to identify potential domain boundaries in the Cas9 protein. A multiple sequence alignment is created by realigning the Cas9 sequence (PF13395) in the PFAM database using MUSCLE, and the alignment is computationally adjusted for diversity and full-length sequence. Sequence conservation is calculated as the relative entropy of observed amino acid frequencies relative to the average frequency of all genes in E. coli. A multi-scale edge filter (a difference of Gaussian (DoG) bandpass filter) is applied to the conservation profile to attribute potential protein domain boundaries, referred to herein as conservation edges. Regions between the conservation edges are selected for deletion of the first round of deletion mutants.

[0011] In one embodiment, the present disclosure describes synthetic NM-Cas9 deletion mutants that are smaller in size but retain activity close to that of the wild-type protein, which can be used to bind DNA in a co-localized complex with a guide RNA in cells to generate double-stranded or single-stranded breaks, or to position an effector group in the vicinity of a target DNA of interest to exert a desired function.

[0012] According to an embodiment, an alignment-based domain detection method is provided to identify regions of DNA-binding proteins, such as Cas9, that are not required for DNA binding and can be removed to form mutant DNA-binding proteins that are smaller in size than wild-type DNA-binding proteins.The methods described herein generate minimized Cas9 variants that exhibit strong activity in bacterial and human cells.According to an embodiment described herein, mutant functional DNA-binding protein variants, such as mutant functional Cas9 variants, are provided that are smaller than wild-type DNA-binding proteins.

[0013] According to certain embodiments, exemplary DNA binding proteins include Cas9 orthologues such as Neisseria meningitides Cas9 (NM, GI:218767588) and Streptococcus thermophilus Cas9 (ST1, GI:116627542), which have been shown to function in both prokaryotes and higher eukaryotes. See Hou, Z. et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proceedings of the National Academy of Sciences of the United States of America 110, 15644-15649, doi:10.1073 / pnas.1313587110 (2013), which is incorporated by reference in its entirety.

[0014] These exemplary Cas9 orthologs have a smaller gene size than Streptococcus pyogenes Cas9 (SP, GI:13622193), i.e., approximately 3200 base pairs versus 4100 base pairs. Thus, aspects of the present disclosure relate to reducing the size of the DNA-binding protein Cas9 such that the efficiency with which it can be delivered is increased, particularly using viral packaging techniques where gene length can greatly affect viral titer. Kumar, M., Keller, B., Makalou, N. & Sutton, RE Systematic determination of the packaging limit of lentiviral vectors. Human gene therapy 12, 1893-1905, doi:10.1089 / 104303401753153947 (2001); Wu, Z., Yang, H. & Colosi, P. Effect of genome size on AAV vector packaging. Molecular therapy : the journal of the American Society of Gene Therapy 18, 80-86, doi:10.1038 / mt.2009.255 (2010); and Gelinas, C. & Temin, HM Nondefective spleen necrosis virus-derived vectors define the upper size limit for packaging reticuloendotheliosis viruses. Proceedings of the National Academy of See Sciences of the United States of America 83, 9211-9215 (1986).

[0015] By synthetically reducing the size of the Cas9 gene, more complex regulatory systems and functional domains can be packaged in a single vector. In another embodiment, a method is provided to synthetically alter PAM specificity, allowing the creation of smaller Cas9 variants with improved targeting capabilities.

[0016] In one embodiment, a method is provided for generating a Cas9 chimera by exchanging the C-terminal domain of a first type of Cas9 with the C-terminal domain of a second type of Cas9. In one embodiment, the present disclosure provides a domain-exchanged Cas9 chimera, such as a functional NM-ST1-Cas9 chimera, by exchanging the C-terminal domain of NM with ST1. The chimeric Cas9 protein exhibits specificity for the guide RNA and PAM of ST1.

[0017] In some embodiments, the cell is a prokaryotic or eukaryotic cell. In some embodiments, the cell is a bacterial cell, a yeast cell, a plant cell, or an animal cell. In some embodiments, the cell is a mammalian cell.

[0018] In one embodiment, the RNA is about 10 to about 500 nucleotides In one embodiment, the RNA is about 20 to about 100 nucleotides.

[0019] In some embodiments, the one or more RNAs are guideRNAs. In some embodiments, the one or more RNAs are crRNAs. In some embodiments, the one or more RNAs are tracrRNAs. In some embodiments, the one or more RNAs are tracrRNA-crRNA fusions.

[0020] In certain embodiments, the target DNA is genomic DNA, mitochondrial DNA, viral DNA, a bindable element, or exogenous DNA.

[0021] In one embodiment, the RNA-guided DNA-binding protein is a RNA-guided DNA-binding protein of the type II CRISPR system that binds to DNA and is guided by one or more RNAs. In one embodiment, the RNA-guided DNA-binding protein is a Cas9 protein that binds to DNA and is guided by one or more RNAs.

[0022] Other features and advantages of particular embodiments of the invention will become more fully apparent in the following description of the embodiments and the drawings thereof, and from the claims.

[0023] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee. The above and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0024] [Figure 1] Figure 1 shows images of E. coli cells transformed with various NM-Cas9 nuclease deletion genes containing a YFP reporter for NM-Cas9 activity. In the absence of NM-Cas9, cells are fluorescent (negative control, top right), and in the presence of full-length nuclease-deleted NM-Cas9, cells are non-fluorescent (positive control, top left). Two of the NM-Cas9 deletions generated are shown, with NM-Cas9-Δ255-449 showing near wild-type levels of repression (bottom left) and NM-Cas9-Δ874-922 showing a near loss of DNA-binding ability (bottom right). [Figure 2-1]Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems, Nucleic Acids Res. 42, 2577-90, which is incorporated by reference in its entirety. Marked in red are sequences used as initial seeds for PFAM realignment. [Figure 2-2] Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems, Nucleic Acids Res. 42, 2577-90, which is incorporated by reference in its entirety. Marked in red are sequences used as initial seeds for PFAM realignment. [Figure 2-3] Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems, Nucleic Acids Res. 42, 2577-90, which is incorporated by reference in its entirety. Marked in red are sequences used as initial seeds for PFAM realignment. [Figure 2-4]Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems, Nucleic Acids Res. 42, 2577-90, which is incorporated by reference in its entirety. Marked in red are sequences used as initial seeds for PFAM realignment. [Diagram 3] FIG. 1 shows the conservation profile of the Cas9 alignment after truncation to the NM-Cas9 position. [Figure 4] FIG. 1 shows the conservation profile truncated to the position in SP-Cas9. [Figure 5A] Figure 1 shows a plot of primary amino acid conservation within the Cas9 protein. The relative entropy has been calculated relative to the average amino acid frequency across all genes in E. coli. The vertical lines above the plot represent the boundaries determined by the alignment-based boundary detection algorithm, and the bold lines represent the six most significant boundaries detected. [Figure 5B] Domain assignments based on Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic acids research 42, 2577-2590, doi:10.1093 / nar / gkt1074 (2014), which is incorporated by reference in its entirety. RuvCI-III is the portion that folds into the RuvC nuclease domain. HNH is the HNH nuclease domain. RRR is the arginine-rich alpha helix region. Shading is an extension of this region based on the arginine-rich stretch around position 140. [Figure 6A]FIG. 1 is a schematic showing the design of the NM Cas9 transcriptional reporter, showing the positions of the protospacer and the NM-specific PAM. [Figure 6B] Figure 1 shows a graph of NM Cas9 transcriptional repression assay data with various nuclease-deficient NM mutants tested in E. coli. Data represent the mean ± standard deviation (n=4). [Figure 6C] Schematic diagram showing the domain structure of NM Cas9. The white box with dashed outline indicates the extent of the largest excision region from the NM mutant that causes little change in DNA binding activity. [Figure 7] Figure 1 shows the design of the NM Cas9 transcriptional reporter for NM Cas9 deletion analysis. The positions of the protospacer and NM-specific PAM are shown. NM Cas9 transcriptional repression assays with various nuclease-deficient NM mutants were tested in E. coli. Data represent the mean ± standard deviation (n=5). [Figure 8A] Figure 1: ST1 Cas9 deletion analysis and functional validation in E. coli and human cells and in particular the design of the ST1 Cas9 transcriptional reporter. The positions of the protospacer and ST1-specific PAM are shown. ST1 Cas9 transcriptional repressor assay with ST1 nuclease-deficient deletion mutants tested in E. coli. Data represent the mean ± standard deviation (n=4). [Figure 8B] Schematic diagram of reporter construct for ST1 activation assay, containing a minimal CMV promoter (min CMV) upstream of the tdTomato reporter. Binding of the ST1 nuclease-deficient Cas9-VP64 fusion protein upstream of the minimal CMV promoter results in transcriptional activation and fluorescence in human cells. [Figure 8C] FIG. 1 shows images of cells transfected with ST1 activators containing deletion mutants, along with sgRNA and a tdTomato reporter, visualized by fluorescence microscopy. [Figure 8D] Graph showing quantification of ST1 activation in FIG. 8C by flow cytometry. Data represent mean ± standard deviation (n=3). [Figure 9] Figure 1. TD Cas9 deletion analysis and functional validation in E. coli. TD nuclease-deficient deletion mutants were tested using a transcriptional repressor assay. Data represent the mean ± standard deviation (n=4). [Figure 10A] FIG. 10: NM-ST1 domain swap analysis measured by transcriptional repression assay, and the design of NM and ST1 transcriptional reporters with the sequences of the NM- or ST1-specific PAMs specifically depicted. [Figure 10B] FIG. 1 is a schematic diagram showing an overview of NM and ST1 Cas9 with the positions of amino acid exchange points. [Figure 10C] Figure 1 shows a graph of fluorescence of NM-ST1 nuclease deletion domain swap mutants co-expressed with NM-Cas9 specific guide RNAs together with a reporter with NM-Cas9 specific PAM sequence. Data represent mean ± standard deviation (n=4). [Figure 10D] Figure 1 shows a graph of fluorescence of NM-ST1 nuclease-deficient domain-swapped mutants co-expressed with NM-Cas9-specific guide RNAs together with a reporter carrying the ST1-Cas9-specific PAM sequence. Data represent the mean ± standard deviation (n=4). [Figure 10E] Figure 1 shows a graph of fluorescence of NM-ST1 nuclease-deficient domain-swapped mutants co-expressed with ST1 Cas9-specific guide RNAs together with a reporter with ST1 Cas9-specific PAM sequence. Data represent mean ± standard deviation (n=4). [Figure 10F] Figure 1 shows a graph of fluorescence of NM-ST1 nuclease-deficient domain-swapped mutants co-expressed with ST1 Cas9-specific guide RNA together with a reporter with NM Cas9-specific PAM sequence. Data represent mean ± standard deviation (n=4). [Figure 11A] FIG. 11. NM-ST1 domain exchange analysis measured by transcriptional repression assay, and the design of an ST1 transcriptional reporter with the sequence of the ST1-specific PAM specifically shown. [Figure 11B]Schematic diagram showing an overview of NM and ST1 Cas9 with the locations of amino acid exchange points. [Figure 11C] Figure 1 shows a graph of fluorescence of NM-ST1 nuclease-deficient domain-swapped mutants co-expressed with ST1 Cas9-specific guide RNAs together with a reporter with ST1 Cas9-specific PAM sequence. Data represent mean ± standard deviation (n=4). [Figure 11D] Figure 1 shows a graph of fluorescence of NM-ST1 nuclease-deficient domain-swapped mutants co-expressed with NM-Cas9-specific guide RNAs together with a reporter carrying the ST1-Cas9-specific PAM sequence. Data represent the mean ± standard deviation (n=4). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention relates to mutant RNA-guided DNA-binding proteins of type II CRISPR system. Such mutants are created by removing less conserved or highly divergent sequences between species in the genus of RNA-guided DNA-binding proteins of type II CRISPR system. According to one aspect, the sequences of species in a family of RNA-guided DNA-binding proteins are aligned to determine low conserved sequences or sequences between conserved edges. These sequences are then deleted from the specific RNA-guided DNA-binding protein. Exemplary RNA-guided DNA-binding proteins include, for example, Cas9 proteins present in type II CRISPR system. Such Cas9 proteins and type II CRISPR systems are well documented in the art. See Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477. The mutant DNA binding proteins described herein can be used to generate double-stranded breaks in target DNA or single-stranded breaks in target DNA, or to bind to target DNA such that an effector group is positioned near the target DNA so that the effector group can interact with the target DNA. Such effector groups include activators, repressors, or epigenetic modifiers known to those skilled in the art.

[0026] Exemplary DNA binding proteins with nuclease activity function to nick or cleave double-stranded DNA. Such nuclease activity can result from DNA binding proteins with one or more polypeptide sequences that exhibit nuclease activity. Such exemplary DNA binding proteins may have two separate nuclease domains, each of which is responsible for cleaving or nicking a specific strand of double-stranded DNA. Exemplary polypeptide sequences with nuclease activity known to those skilled in the art include McrA-HNH nuclease-associated domain and RuvC-like nuclease domain. Thus, exemplary DNA binding proteins are naturally occurring proteins that include one or more of McrA-HNH nuclease-associated domain and RuvC-like nuclease domain.

[0027] According to one embodiment, the DNA binding protein having two or more nuclease domains may be modified or altered so that all but one of the nuclease domains are inactivated.Such modified or altered DNA binding protein is called DNA binding protein nickase, as long as the DNA binding protein only cuts or forms a nick on one strand of double-stranded DNA.When it is guided to DNA by RNA, the DNA binding protein nickase is called RNA-guided DNA binding protein nickase.

[0028] An exemplary DNA binding protein is the RNA-guided DNA binding protein of type II CRISPR system.An exemplary DNA binding protein is Cas9 protein.

[0029] In S. pyogenes, Cas9 creates a blunt-ended double-stranded break 3 bp upstream of the protospacer adjacent motif (PAM) by a process mediated by two catalytic domains in the protein: an HNH domain that cleaves the complementary strand of DNA and a RuvC-like domain that cleaves the non-complementary strand. See Jinke et al., Science 337, 816-821 (2012), which is incorporated by reference in its entirety. Cas9 proteins are known to be present in a number of type II CRISPR systems, including the following, which are reviewed in supplementary information in Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477: Methanococcus maripaludis strain C7; Corynebacterium diphtheriae; Corynebacterium efficiens strain YS-314; Corynebacterium glutamicum ATCC13032 Kitasato strain; Corynebacterium glutamicum ATCC13032 Bielefeld strain; Corynebacterium glutanicum strain R; Corynebacterium kroppenstedtii strain DSM44385; Mycobacterium abscessus strain ATCC19977; Nocardia farcinica farcinica strain IFM10152; Rhodococcus erythropolis strain PR4; Rhodococcus jostii strain RHA1; Rhodococcus opacus strain B4 uid36573; Acidothermus cellulolyticus strain 11B;Arthrobacter chlorophenolicus strain A6; Kribbella flavida strain DSM17836 uid43465; Thermomonospora curvata strain DSM43183; Bifidobacterium dentium strain Bd1; Bifidobacterium longum strain DJO10A; Slackia heliotrinireducens strain DSM20476; Persephonella marina strain EX H1; Bacteroides fragilis strain NCTC9434; Capnocytophaga ochracea strain DSM7271;Flavobacterium psychrophilum strain JIP02 86;Akkermansia muciniphila strain ATCC BAA835;Roseiflexus castenholzii strain DSM13941;Roseiflexus strain RS1;Synechocystis strain PCC6803;Elusimicrobium minutum strain Pei191;Uncultured Termite group 1 bacterium phylotype Rs D17;Fibrobacter succinogenes strain S85;Bacillus cereus cereus ATCC10987 strain; Listeria innocua; Lactobacillus casei; Lactobacillus rhamnosus GG strain; Lactobacillus salivarius UCC118 strain;Streptococcus agalactiae strain A909; Streptococcus agalactiae strain NEM316; Streptococcus agalactiae strain 2603; Streptococcus dysgalactiae equisimilis strain GGS124; Streptococcus equi subspecies zooepidemicus strain MGCS10565; Streptococcus gallolyticus strain UCN34 uid46061; Streptococcus gordonii Challis strain CH1 substrain; Streptococcus mutans mutans NN2025 uid46353;Streptococcus mutans;Streptococcus pyogenes M1 GAS;Streptococcus pyogenes MGAS5005;Streptococcus pyogenes MGAS2096;Streptococcus pyogenes MGAS9429;Streptococcus pyogenes MGAS10270;Streptococcus pyogenes MGAS6180;Streptococcus pyogenes MGAS315;Streptococcus pyogenes SSI-1;Streptococcus pyogenes MGAS10750;Streptococcus pyogenes NZ131 strain; Streptococcus thermophiles strain CNRZ1066; Streptococcus thermophiles strain LMD-9; Streptococcus thermophilus strain LMG18311; Clostridium botulinum strain A3 Loch Maree; Clostridium botulinum strain B Eklund 17B; Clostridium botulinum strain Ba4 657; Clostridium botulinum strain F Langeland;Clostridium cellulolyticum strain H10; Finegoldia magna strain ATCC29328; Eubacterium rectale strain ATCC33656; Mycoplasma gallisepticum; Mycoplasma mobile strain 163K; Mycoplasma penetrans; Mycoplasma synoviae strain 53; Streptobacillus moniliformis strain DSM12112; Bradyrhizobium strain BTAi1; Nitrobacter hamburgensis hamburgensis strain X14;Rhodopseudomonas palustris BisB18;Rhodopseudomonas palustris strain BisB5;Parvibaculum lavamentivorans strain DS-1;Dinoroseobacter shibae strain DFL12;Gluconacetobacter diazotrophicus Pal 5 strain FAPERJ;Gluconacetobacter diazotrophicus Pal 5 strain JGI;Azospirillum strain B510 uid46085;Rhodospirillum rubrum strain ATCC11170;Diaphorobacter TPSY strain uid29975; Verminephrobacter eiseniae strain EF01‐2; Neisseria meningitides strain 053442; Neisseria meningitides alpha14; Neisseria meningitides strain Z2491;Desulfovibrio salexigens strain DSM 2638;Campylobacter jejuni doylei strain 269 97;Campylobacter jejuni strain 81116;Campylobacter jejuni;Campylobacter lari strain RM2100;Helicobacter hepaticus;Wolinella succinogenes;Tolumonas auensis strain DSM 9187;Pseudoalteromonas atlantica strain T6c;Shewanella pareana pealeana strain ATCC700345;Legionella pneumophila strain Paris;Actinobacillus succinogenes strain 130Z;Pasteurella multocida;Francisella tularensis novicida strain U112;Francisella tularensis subsp. holarctica;Francisella tularensis strain FSC 198;Francisella tularensis subsp. tularensis;Francisella tularensis strain WY96‐3418;and Treponema denticola ATCC 35405 strain. Thus, embodiments of the present disclosure relate to variants of Cas9 proteins present in type II CRISPR systems, such as any one of the species identified above. Exemplary Cas9 proteins are those found in Neisseria meningitidis, such as Neisseria meningitidis strain 053442, Neisseria meningitidis strain alpha14, Neisseria meningitidis strain Z2491;

[0030] The cells of the present disclosure include any cell into which an exogenous nucleic acid can be introduced and expressed as described herein. It should be understood that the basic concepts of the present disclosure described herein are not limited by cell type. The cells of the present disclosure include eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, archaeal cells, eubacterial cells, and the like. The cells include eukaryotic cells, such as yeast cells, plant cells, and animal cells. Particular cells include mammalian cells. Particular cells include stem cells, such as pluripotent stem cells, such as human induced pluripotent stem cells.

[0031] Target nucleic acids include any nucleic acid sequence that mutant RNA-guided DNA-binding protein nucleases may be useful for nicking or cleaving. Target nucleic acids include genes. For the purposes of this disclosure, DNA, such as double-stranded DNA, may comprise the target nucleic acid, and the colocalization complex may bind to or colocalize with DNA at, adjacent to, or near the target nucleic acid, such that the colocalization complex may have a desired effect on the target nucleic acid. Such target nucleic acids may include endogenous (or natural) nucleic acids and exogenous (or foreign) nucleic acids. Based on this disclosure, a person skilled in the art can easily identify or design guide RNAs and mutant Cas9 proteins that colocalize with DNA that comprises the target nucleic acid. DNA includes genomic DNA, mitochondrial DNA, viral DNA, binding elements, or foreign DNA.

[0032] Exogenous nucleic acid (i.e., not part of the cell's natural nucleic acid composition) may be introduced into a cell using any method of introduction known to those of skill in the art, including gene transfer, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation, and the like. Those of skill in the art will readily understand and apply such methods using readily identifiable literature sources.

[0033] In one embodiment, the genetic material required to encode the Cas9 protein is reduced by deleting portions of the Cas9 protein that are less conserved or diverged within species in the Cas9 family, or portions of the Cas9 protein that are between the edges of conservation within species in the Cas9 family. By reducing the size of the nucleic acid required to encode a functional Cas9, the vector designed to deliver Cas9 can include additional nucleic acids, such as nucleic acids encoding guide RNAs or regulatory elements or effector domains. When using the smallest characterized Cas9 family member, approximately 4,500 kilobases of DNA are required to encode the genetic elements (Cas9 protein and gRNA) required to properly localize Cas9 to the desired genomic locus. At approximately 4,500 base pairs, Cas9 is close to the size limit for packaging into adeno-associated virus (AAV)-based viral vectors, which are the viral vectors that have regulatory approval in Europe. Furthermore, some of the first transcriptional and epigenetic effector domains fused to programmable DNA-binding proteins are larger than 2,000 base pairs, which, when fused to Cas9, far exceeds the packaging limit of AAV vectors and approaches the limit of lentiviral packaging systems (approximately 8,000 base pairs).

[0034] The following examples are provided as representative of the present disclosure, and should not be construed as limiting the scope of the disclosure, as these examples and other equivalent embodiments will become apparent in light of the present disclosure, the drawings, and the appended claims. EXAMPLES

[0035] Example I Cas9 mutants To overcome the problem of the large size of the gene encoding Cas9, we performed targeted deletions of various regions in NM-Cas9 (Neisseria meningitidis Cas9), one of the smallest characterized Cas9 family members. The size of NM-Cas9 is 3,249 bp. The requirements for genome targeting and residues involved in nuclease activity were determined. To generate a smaller NM-Cas9, an alignment of Cas9 proteins was generated and low-conserved adjacent sections or sections between conserved edges were identified for deletion. Multiple regions of interest were identified and selectively removed from NM-Cas9, and then their function was assessed using a Cas9 suppressor assay. The assay used an NM-Cas9 mutant that lacks nuclease activity but can target the 5′ region of a reporter gene. When NM-Cas9 is able to bind to the reporter gene, it suppresses transcription, and in the case of a fluorescent reporter, cells become non-fluorescent.

[0036] Cas9 alignment and deletion prediction: Full-length sequences of Cas9 homologs were obtained from database searches such as the PFAM database or jackHMMER (RD Finn, J. Clements, SR Eddy, Nucleic Acids Research (2011) Web Server Issue 39:W29-W37, which is incorporated by reference in its entirety). If the collection of sequences was not aligned, an alignment was created using an alignment algorithm such as CLUSTALW (Sievers F, Wilm A, Dineen DG, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG (2011), which is incorporated by reference in its entirety) or equivalent. The alignment was computationally truncated to the position of the sequence of interest and adjusted to make the alignment less biased (e.g., sequences with >95% pairwise identity were removed). The degree of conservation is calculated as the entropy or relative entropy of amino acid frequency per position, taking into account the amount of amino acids and gaps at that position. Regions of low conservation or regions between conserved edges are targeted for deletion. Experimental validation is repeated to extend or shift the deletion.

[0037] Construction and Characterization of Deletions: Bacterial plasmids expressing nuclease-deficient NM-Cas9 were previously generated as described in Esvelt, KM, Mali, P., Braff, JL, Moosburner, M., Yaung, SJ, and Church, GM (2013) Nat Methods 10, 1116-1121, which is incorporated by reference in its entirety. To generate targeted deletions in NM-Cas9, Gibson assembly was used as described in Gibson, DG, Young, L., Chuang, RY, Venter, JC, Hutchison, CA, 3rd, and Smith, HO (2009) Nat Methods 6, 343-345, which is incorporated by reference in its entirety. Primers containing overlapping complementary portions and designed to remove the targeted region in NM-Cas9 and insert a SGGGS linker were purchased and used in PCR reactions. PCR fragments were gel purified, assembled in vitro using Gibson assembly, and transformed into E. coli. The sequences of clones were verified, and clones were tested using a modified version of a pre-made NM-Cas9 reporter plasmid (see (2013) Nat Methods 10, 1116-1121) in which a single (rather than two) plasmid contains the NM-Cas9 spacer, the target protospacer, and a YFP reporter for NM-Cas9 activity. Briefly, in this assay, cells are co-transformed with a synthetic NM-Cas9 variant and a reporter plasmid. The doubly transformed cells are then grown at 37°C, and the amount of YFP fluorescence is measured using a fluorescent plate reader and compared to cells transformed with a control plasmid carrying wild-type nuclease-deficient NM-Cas9 and the reporter plasmid.

[0038] Below are the sequences of the two largest NM-Cas9 single deletion mutants that retained near wild-type levels of activity as measured by the YFP reporter assay. The sequence of the SGGGS linker replacing the deleted region in NM-cas9 is shown in uppercase.

[0039] [ka]

[0040] [ka]

[0041] [ka] [ka]

[0042] According to the methods described herein, multiple deletions within NM-Cas9 have been identified. The largest, NM-Cas9-Δ255-449, removes 595 base pairs, resulting in only a 16% reduction in activity as measured by a reporter assay. In some embodiments, mutant Cas9 proteins are provided that contain 1000 or 900 base pairs less than wild-type Cas9, such as NM-Cas9, and retain near wild-type levels of activity.

[0043] Example II Targeting the Cas9 nuclease domain for deletion If a nickase- or nuclease-deficient allele of Cas9 is desired, the Cas9 nuclease domain can be targeted for deletion along with the surrounding nucleotides, along with targeting regions of low sequence conservation or between the sequence-conserved edges. Using such an approach, we generated a functional NM-Cas9 allele, NM-Cas9-Δ567-654, lacking the HNH motif and surrounding nucleotides. This allele retained nearly the same DNA-binding ability as the wild type, as measured by a YFP reporter assay.

[0044] Example III A method for constructing unbiased NM-Cas9 deletion libraries In addition to targeted approaches to create Cas9 deletions, embodiments of the present disclosure include high-throughput approaches for random deletion generation and screening of functional mutants. According to an exemplary method, plasmid DNA containing the desired Cas9 allele may be sheared using promiscuous nucleases, sonication, repeated pipetting of samples, or other chemical, enzymatic, or environmental means. After fragmentation, the plasmid DNA may be treated with an exonuclease to remove nucleotides from the Cas9 gene. After exonuclease treatment, the fragmented ends are blunted with an enzyme such as mung bean nuclease or Klenow polymerase and ligated together to regenerate the Cas9 plasmid containing the random deletion. To insert a foreign domain, such as a linker or effector motif, into the deleted portion of Cas9, such a domain may be ligated to the blunt-ended fragment DNA, and then circularization of the plasmid results in a Cas9 coding sequence with the foreign domain inserted into the deleted portion of Cas9. The library of circularized molecules is then transformed into E. coli and plasmid DNA is extracted.

[0045] At this point, the library may be transformed into cells that contain a reporter assay for Cas9 activity, and library members that maintain functional activity may be identified. Alternatively, to reduce the size of the library to be screened, the coding sequence of Cas9 from the newly created library may be isolated by digestion or PCR, and the fragments may be size-selected to be shorter than the original wild-type Cas9 gene. These smaller members may then be religated into the original vector and transformed into cells that contain a reporter for Cas9 activity.

[0046] In addition to shearing the plasmid, a library of oligonucleotides can be generated that have 3′ homology to the Cas9 gene but 5′ homology to each other, with the 3′ end of each oligonucleotide binding to a different section of Cas9 of about 30 base pairs. These oligonucleotides cover both the sense and antisense strands of the Cas9 coding sequence. These oligonucleotides can then be used to perform PCR to generate a series of Cas9 fragments, where each product of a given sense PCR reaction is complementary to all of the other antisense PCR products, and vice versa. These fragments can then be annealed together using methods such as Gibson assembly or overlap extension PCR, followed by ligation into a vector backbone and transformation into cells to generate a library of Cas9 variants in which random sections of the Cas9 gene have been removed. For longer linkers or to insert an effector domain within the deleted region, the oligonucleotide should have complementarity to the longer linker or effector domain at its 5' end, and this domain should then be introduced during the Gibson assembly reaction or overlap extension PCR.Once the library is generated, functional variants can be identified using a reporter assay, such as the YFP reporter system described herein.

[0047] Example IV Vector construction Cas9 nuclease deletion plasmids were constructed from ST1 (Addgene #48659) or NM and TD plasmids (Addgene #48646 and 48648, respectively) by introducing the following point mutations (NM: D16A D587A H588A N611A, and TD: D13A D878A H879A N902A). Gibson assembly was used to generate Cas9 deletions. When generating internal deletions, the internal deletions were linked with a 5 amino acid Ser‐Gly‐Gly‐Gly‐Ser linker, except for NMΔ566‐620, which lacks a linker between the linked fragments. An N-terminal domain swap fused residues 1–117 of ST1 to residues 118–1082 of NM. A C-terminal domain swap fused residues 1–727 of NM to residues 743–1121 of ST1.

[0048] Example V Bacterial reporter constructs The reporter construct used for the analysis of deletion mutants is similar to that previously published, except that it combines the spacer element and the YFP reporter in a single SC101-kanR plasmid backbone. The reporter construct for domain swapping analysis is the same as that used previously. See Esvelt, KM et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature methods 10, 1116-1121, doi:10.1038 / nmeth.2681 (2013), which is incorporated by reference in its entirety.

[0049] Example VI Mammalian reporter constructs The M-ST1n-VP64 construct, ST1 guide RNA plasmid, and ST1-specific mammalian transcriptional reporter were previously published in Esvelt, KM et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature methods 10, 1116-1121, doi:10.1038 / nmeth.2681 (2013) (Addgene#48675, 48672, and 48678, respectively). Deletion mutants were generated as for the bacterial constructs.

[0050] Example VII Suppression assay Cas9 repression assays were performed by co-transforming NEB 10-beta cells (New England NioLabs) with the appropriate spacer / reporter construct and Cas9 vector to be tested. Colonies resulting from transformation were picked and grown in 96-well plates at 37°C with continuous shaking. Plates were read the next day using a Synergy Neo microplate reader (BioTek) to measure fluorescence at 495-528 nm and absorbance at 600 nm. In the swapping experiments, two different spacer / protospacer combinations (A and B) previously published (see Esvelt, KM et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature methods 10, 1116-1121, doi:10.1038 / nmeth.2681 (2013)) were tested. In all other experiments, only spacer / protospacer combination B was examined.

[0051] Example VIII Cell culture and transfection HEK 293T cells were maintained in high glucose Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and penicillin / streptomycin (Invitrogen). Cells were maintained at 37°C and 5% CO in a humidified incubator. 2 Cells were transfected in 24-well plates seeded at 50,000 cells per well. 2.5 μl of Lipofectamine 2000 was used to deliver 400 ng of Cas9 activator, 100 ng of gRNA, and 60 ng of reporter plasmid to each well. Cells were grown for an additional 36–48 h and then assayed using immunofluorescence or FACS.

[0052] Example IX Multiple sequence alignment and edge filters A multiple sequence alignment was generated by realigning Cas9 sequences from the PFAM database (PF13395, 798 sequences) in MUSCLE and adjusting the alignment for diversity and full length sequences using a MATLAB script. This method, which is described in more detail below, resulted in 217 sequences.

[0053] To obtain the final realignment of Cas9 sequences in the PFAM database (PF13395, 798 sequences), the following steps were performed (all program codes in MATLAB): We obtained the alignment from Fonfara I, et al., (2014) Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems, Nucleic Acids Res. 42, 2577-90, and included only sequences from groups IIA and IIC, and from the latter only sequences up to the branch separating IIA and IIC (marked with a red box in Figure 2 (Figure 2-1 to Figure 2-4)) for a total of 49 sequences.

[0054] The sequences were then divided into two groups, those with and those without a large insertion around position 150 (which distinguishes, for example, NM-Cas9 from ST-Cas9 on the one hand, and SP-Cas9 from TD-Cas9 on the other). These groups were aligned separately using MUSCLE (see Edgar, RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Res 32, 1792-97) and realigned using a windowed approach (due to the length of the sequences) before cross-profile alignment into a single seed alignment.

[0055] The sequences in PF13395 were realigned using seed alignment in MUSCLE. All alignments using the seed are performed by aligning each target sequence to the seed one by one. This alignment is used to determine the top hit identity between the seed and the target sequence, and then sorted again in descending order of top hit identity. The target sequences are then realigned to the seed one by one, this time in descending order of identity. This two-step approach ensures the robustness of the alignment. Furthermore, these sequences are separated according to whether they contain insertions or not, and the two separate groups are realigned to the seed as profiles. Short sequences and sequences with large truncations are manually removed. Sequences with more than 90% pairwise similarity are removed.

[0056] The two resulting alignments were aligned to each other across profiles, yielding a total of 217 sequences. The resulting alignment was then truncated to the position of the Cas9 ortholog of interest.

[0057] Sequence conservation was calculated as the relative entropy to the average amino acid background frequency of all genes in E. coli O157. Domain boundary detection was performed by applying a Difference of Gaussians (DoG) edge filter (see Marr, D. & Hildreth, E. Theory of edge detection. Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society 207, 187-217 (1980), which is incorporated by reference in its entirety) to the resulting conservation profile, averaging over multiple length scales to achieve robustness to parameter selection, and detection at various length scales.

[0058] Specifically, the degree of conservation of the alignment was calculated as the relative entropy of the amino acid frequency to the average frequency of all genes of E. coli O157 (see Cover, TM and Thomas, JT (2006) Elements of Information Theory; 2nd edition, Wiley-Interscience).

[0059]

number

[0060] In the formula, p i a is the frequency of amino acid a at position i, and q a is the average frequency of amino acid a. It is the sum of all 20 amino acids. The log is base 2 and the entropy is given in bits. The average frequency q a is as follows. {ACDEFGHIKLMNPQRSTVWY} = 0.094 0.012 0.052 0.058 0.038 0.073 0.022 0.059 0.045 0.104 0.027 0.041 0.044 0.044 0.057 0.060 0.055 0.070 0.015 0.029. After truncation to the NM-Cas9 position, alignment of Cas9 gives the conservation profile in Figure 3.

[0061] The conservation profile is plotted abbreviated to the position in SP-Cas9 (an example of a larger Cas9 protein of the Type IIA subfamily). Similar features are observed N-terminally to approximately NM145 (SP170) and further after NM200 (SP400), which is the large insertion previously described. See FIG. 4.

[0062] Potential domain boundaries were identified by applying a multi-scale edge filter to the conservation profiles, which calculates Difference of Gaussians (DoG) (see Marr, D and Hildreth, E (1980) Theory of Edge Detection, Proc R Soc Lond B Biol Sci 207, 187-217, which is incorporated by reference in its entirety) at various scales and sums the resulting graph. The extremes of this curve are interpreted as boundaries between low and high conserved regions in the protein. These domains may show different levels of conservation due to potentially different functional importance leading to different rates of evolutionary divergence. Different degrees of conservation may be characteristic of multi-domain proteins resulting from domain insertions over evolutionary time. The extreme values ​​are used to rank the boundaries in terms of importance.

[0063] In a Cas9 alignment limited to the NM position, the following boundary or conserved edge or edge amino acids were identified using the methods described herein:

[0064] [Table 1]

[0065] Example X Computational analysis of Cas9 family members To identify regions within the multidomain Cas9 protein that may be suitable for deletion, a bioinformatics approach was used to identify potential boundaries between domains. The full-length Cas9 sequence (PF13395) from PFAM was realigned using a well-curated seed alignment as previously described, and sequences with high pairwise identity were removed. Primary sequence conservation was calculated as the relative entropy of the observed amino acid frequency relative to the average frequency across all genes in E. coli (see Cover, TM, Thomas, JT Elements of Information Theory, 2nd edition. (Wiley-Interscience, 2006)). Since domains in a multidomain protein can be expected to show different levels of sequence conservation, applying a multiscale edge filter to the conservation profile may be suitable to identify the location of domain boundaries.

[0066] Edge detection was performed on the conservation profile using a Difference of Gaussians (DoG) bandpass filter, which is sensitive to a narrow range of spatial frequencies. See Marr, D. & Hildreth, E. Theory of edge detection. Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society 207, 187-217 (1980). Averaging over multiple scales (5-50 amino acids) was performed to allow detection at different length scales and to make the filter insensitive to specific parameter choices. The bandpass filter was then applied to the conservation profile of the Cas9 alignment. The identified potential boundary locations for NM-Cas9 are shown in Figure 5A. The top six boundaries are shown as long, thick red lines. As shown in the figure, the filter correctly identifies the arrangement of known HNH and RuvC domains beginning upstream of position 500 and extending to position 750 as previously assigned in Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR / Cas system provides immunity in Escherichia coli. Nucleic acids research 39, 9275-9282, doi:10.1093 / nar / gkr606 (2011) and Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic acids research 42, 2577-2590, doi:10.1093 / nar / gkt1074 (2014), each of which is incorporated by reference in its entirety (see Figure 5B). The interdomain boundaries around positions 560 and 620 are also accurately predicted.The boundary of the first arginine-rich α-helix at position 88 is also predicted. One of the higher boundaries on the N-terminal side of the protein, at position 144, was not previously identified as a domain boundary in Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic acids research 42, 2577-2590, doi:10.1093 / nar / gkt1074 (2014), but it further delineates the arginine (Arg)-rich α-helical region and now contains a second conserved arginine-rich helix.

[0067] Example XI Cas9 truncation and deletion analysis To experimentally explore functional and potentially non-functional domains within Cas9, we generated N- and C-terminal truncations as well as a series of moderate internal deletions based on the domain discovery analysis described herein. The effects of deletions were analyzed using a transcriptional repressor assay in E. coli, in which a functional nuclease-deficient Cas9 protein binds to the 5' end of a YFP reporter, thereby reducing reporter expression levels (see Figure 6A). See Qi, LS et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173-1183, doi:10.1016 / j.cell.2013.02.022 (2013); Esvelt, KM et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature methods 10, 1116-1121, doi:10.1038 / nmeth.2681 (2013); and Bikard, D. et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic acids research 41, 7429-7437, doi:10.1093 / nar / gkt520 (2013), each of which is incorporated by reference in its entirety. While none of the N- or C-terminal truncations were able to repress the reporter, two internal deletions downstream and upstream of the 288 boundary, NMΔ255-289 and NMΔ330-389, as well as the NMΔ566-620 deletion, showed repression close to wild-type levels (Fig. 6B and Fig. 7). Multiple rounds of iteratively expanding deletions were further analyzed to assay their ability to repress the reporter (Fig. 6B).Two large non-overlapping regions were identified (254-449 and 567-654, constituting 18% and 8% of the total length of the protein, respectively) that could be removed without significant loss of NM activity (Figure 6C). As shown in the alignment, NM 254-449 is a relatively low-conserved stretch within the protein region specific to the Cas9 protein. Positions 567 to 654 are the HNH domain, a domain known to be important for Cas9 DNA catalysis but dispensable for DNA binding.

[0068] To demonstrate that the region removed from NM-Cas9 is not specific to NM but is a general region that can be removed from other Cas9 family members, we made corresponding deletions in nuclease-deficient variants of Streptococcus thermophilus Cas9 (ST1) and Treponema denticola Cas9 (TD, GI:42525843) and measured their function using transcriptional repression assays (Figures 8A and 9). The corresponding deletion mutants in both ST1 and TD showed similar activity to their wild-type counterparts, suggesting that the removed region is dispensable for Cas9 DNA binding across the Cas9 phylogenetic tree and among more distant members of the type II-A subfamily, such as TD.

[0069] The activity of the two largest functional deletions described herein was tested in ST1-Cas9 using a transcriptional activator assay (Figure 8B). Consistent with the analysis in E. coli, both ST1 deletion mutants retained activity comparable to the wild-type protein when fused to the VP64 activation domain and targeted to a fluorescent reporter in human cells (Figures 8C-D). The larger of the two deletion mutants, Δ255-450 (ST1 numbering), results in a Cas9 gene that is 2,793 base pairs in size.

[0070] Example XII Cas9 domain exchange The N- and C-terminal domains of Cas9 may play important roles in crRNA:tracrRNA binding and / or PAM selectivity. To analyze activity, we generated a series of domain-swapped mutants between NM and ST1, in which the N- and / or C-termini of NM were replaced with homologous regions of ST1. We then altered the guide RNA and / or Cas9-specific PAM in the reporter and tested the chimeric proteins using the transcriptional reporter assay described herein to determine the impact of the domain swap on protein specificity (Figure 10A). The exact location of the domain swap was determined based on domain boundary analysis; that is, we selected positions that were as close as possible to the most significant N- and C-terminal boundaries identified (Figure 5A) and at the same time almost perfectly conserved within the alignment (Figure 10B). None of the N-terminal domain swaps between NM and ST1 conferred novel properties to NM, suggesting that the N-terminus of ST1 is not modular and functions in conjunction with other regions of ST1 that were not introduced (Figures 10C-10F). C-terminal exchange generated NM-ST1 hybrids that could interact with ST1 crRNA:tracrRNA complexes and further silence reporters with ST1-specific PAMs (Figure S10E). This result was further validated with other ST1-specific reporters, as shown in Figures S11(A)-S11(D). Aspects of the invention include the following. Appendix 1 identifying a first amino acid sequence that is a highly conserved amino acid sequence adjacent to two adjacent amino acid sequences that are less conserved, or a less conserved amino acid sequence adjacent to two adjacent amino acid sequences that are highly conserved, within a family of DNA binding proteins; identifying a nucleic acid sequence corresponding to said first amino acid sequence; generating a nucleic acid sequence for a target DNA binding protein that lacks a nucleic acid sequence corresponding to said first amino acid sequence; and generating a mutant DNA binding protein from said generated nucleic acid sequence; A method for producing a mutant DNA binding protein comprising: Appendix 2 2. The method of claim 1, wherein the target DNA binding protein has nuclease activity. Appendix 3 2. The method of claim 1, wherein the target DNA binding protein is nickase. Appendix 4 2. The method of claim 1, wherein the target DNA binding protein is nuclease-deficient. Appendix 5 2. The method of claim 1, wherein a nucleic acid sequence corresponding to the first amino acid sequence is deleted from a nucleic acid sequence for the target DNA binding protein. Appendix 6 The method of claim 1, wherein a nucleic acid sequence corresponding to the first amino acid sequence is deleted from a nucleic acid sequence for the target DNA binding protein and replaced with a linker. Appendix 7 2. The method of claim 1, wherein a nucleic acid sequence corresponding to the first amino acid sequence is deleted from a nucleic acid sequence for the target DNA binding protein and replaced with a SGGGS linker. Appendix 8 2. The method of claim 1, wherein the DNA binding protein is a Cas9 protein. Appendix 9 2. The method of claim 1, wherein the first amino acid sequence has a pair of edge amino acid sequences that distinguish the first amino acid sequence from an adjacent amino acid sequence. Appendix 10 A mutated Cas9 protein of sequence ID No. 1. Appendix 11 A mutated Cas9 protein of sequence ID No. 2. Appendix 12 Identifying one or more amino acid sequences within the family of Cas9 proteins that are not highly conserved or are less conserved; identifying a nucleic acid sequence corresponding to said one or more identified amino acid sequences; generating a nucleic acid sequence for a target Cas9 protein that lacks a nucleic acid sequence corresponding to said one or more identified amino acid sequences; and generating a mutant Cas9 protein from the generated nucleic acid sequence. A method for producing a mutant Cas9 protein, comprising: Appendix 13 13. The method of claim 12, wherein the target Cas9 protein has nuclease activity. Appendix 14 13. The method of claim 12, wherein the target Cas9 protein is nickase. Appendix 15 13. The method of claim 12, wherein the target Cas9 protein is nuclease-deficient. Appendix 16 13. The method of claim 12, wherein a nucleic acid sequence corresponding to the one or more identified amino acid sequences is deleted from a nucleic acid sequence for the target Cas9 protein. Appendix 17 13. The method of claim 12, wherein a nucleic acid sequence corresponding to the one or more identified amino acid sequences is deleted from the nucleic acid sequence for the target Cas9 protein and replaced with a linker. Appendix 18 13. The method of claim 12, wherein a nucleic acid sequence corresponding to the one or more identified amino acid sequences is deleted from the nucleic acid sequence for the target Cas9 protein and replaced with a SGGGS linker. Appendix 19 Replacing the C-terminal domain of a first Cas9 protein species with the C-terminal domain of a second Cas9 protein species. A method for producing a mutant Cas9 protein, comprising: Appendix 20 20. The method of claim 19, wherein the first Cas9 protein species is NM-Cas9 and the second Cas9 protein species is ST1-Cas9. Appendix 21 Replacing the N-terminal domain of a first Cas9 protein species with the N-terminal domain of a second Cas9 protein species. A method for producing a chimeric Cas9 protein, comprising: Appendix 22 22. The method of claim 21, wherein the first Cas9 protein species is NM-Cas9 and the second Cas9 protein species is ST1-Cas9.

Claims

1. A Cas9 (NM-Cas9) protein of Neisseria meningitides, which has a deletion of 35 or more amino acids in a region of positions 254-449 of the Cas9 protein of Neisseria meningitides compared to a wild-type Cas9 protein, It has RNA-guided DNA binding activity. NM-Cas9 deletion mutant Cas9 modified protein.

2. A Streptococcus thermophilus Cas9 (ST1-Cas9) protein having a deletion of 35 or more amino acids in a region between positions 255 and 450 of the protein, compared to a wild-type Cas9 protein; It has RNA-guided DNA binding activity. ST1-Cas9 deletion mutant Cas9 modified protein.

3. A deletion mutant Cas9 modified protein described in claim 1 or claim 2, which has nuclease activity.

4. A deletion mutant Cas9 modified protein described in claim 1 or claim 2, having nickase activity.

5. A deletion mutant Cas9 modified protein described in claim 1 or claim 2, which is nuclease deficient.

6. A deletion mutant Cas9 modified protein as described in claim 1 or claim 2, comprising one or more SGGGS linkers.

Images