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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
  • G01N2333/922—Ribonucleases (RNAses); Deoxyribonucleases (DNAses)

Definitions

• CRISPR RNA crRNA fused to a normally trans-encoded tracrRNA (“trans-activating CRISPR RNA”) is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA.
• trans-activating CRISPR RNA a normally trans-encoded tracrRNA fused to a normally trans-encoded tracrRNA
• Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA.
• gRNA homologous to a target site results in Cas9 recruitment and degradation of the target DNA. See H. Deveau et al, Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology 190, 1390 (Feb, 2008).
• Cas9 is a DNA nuclease that can be programmed to target nearly any region of a genome by expressing a guide RNA (gRNA) that contains a motif that recruits Cas9 and 20 basepairs of complementarity to a region of the genome where targeting is desired.
• gRNA guide RNA
• All characterized and putative Cas9 family members are several kilobases in size (>3,000 basepairs) with the smallest functionally validated member NM-Cas9 (Neisseria meningitides Cas9) being 3,249 basepairs in size. The large size of this protein limits its potential for biotechnology and therapeutic applications due to difficulties of delivery and manipulation.
• RNA guided DNA binding protein of a Type II CRISPR System is a Cas9 protein.
• aspects of the present disclosure are directed to an RNA guided DNA binding protein of a Type II CRISPR System which has been engineered to omit portions of the protein while still functioning as an RNA guided DNA binding nickase that can bind to target DNA and create a single stranded break or nick in target DNA.
• the RNA guided DNA binding protein of a Type II CRISPR System is a Cas9 protein.
• aspects of the present disclosure are directed to an RNA guided DNA binding protein of a Type II CRISPR System which has been engineered to omit portions of the protein while still functioning as an RNA guided DNA binding protein which is nuclease null, that is, the RNA guided DNA binding protein lacks nuclease activity.
• the RNA guided DNA binding protein of a Type II CRISPR System is a Cas9 protein.
• portions of an RNA guided DNA binding protein are identified for deletion by identifying within a population of species of the RNA guided DNA binding protein sequences which are not well conserved or are otherwise highly divergent within a particular RNA guided DNA binding protein family and/or protein sequences between boundaries between low and high conservation referred to herein as "conservation edges" within a particular RNA guided DNA binding protein family.
• amino acid sequences within a DNA binding protein such as an RNA guided DNA binding protein, such as Cas9, are identified as having either high conservation or low conservation using methods described herein and as are known to those of skill in the art.
• amino acid sequences of high conservation and amino acid sequences of low conservation are adjacent, such as immediately adjacent, to one another within the protein sequence of the DNA binding protein as a whole.
• the amino acid sequences of high conservation and the amino acid sequences of low conservation are distinguished by an amino acid which separates an amino acid sequence of high conservation from an amino acid sequence of low conservation.
• the amino acid which separates an amino acid sequence of high conservation from an amino acid sequence of low conservation is referred to herein as an "edge amino acid” or a “conservation edge” to the extent that it is at an edge or terminal portion of either an amino acid sequence of high conservation or an amino acid sequence of low conservation.
• the methods of the present disclosure contemplate identifying an amino acid which separates an amino acid sequence of high conservation from an amino acid sequence of low conservation or otherwise distinguishes an amino acid sequence of high conservation from an amino acid sequence of low conservation.
• an amino acid is referred to herein as an "edge amino acid.”
• a pair of edge amino acids may flank or bound on either end an amino acid sequence of high conservation.
• a pair of edge amino acids may flank or bound on either end an amino acid sequence of low conservation.
• one exemplary embodiment relates to the identification within the protein sequence of a DNA binding protein as a whole, adjacent amino acid sequences of high conservation and amino acid sequences of low conservation.
• one exemplary embodiment relates to the identification within the protein sequence of a DNA binding protein as a whole, sequences of high conservation in tandem or in series with sequences of low conservation, and in particular, a sequence of high conservation (HC) bounded on either end by a sequence of low conservation (LC) or alternatively a sequence of low conservation (LC) bounded on either end by a sequence of high conservation (HC).
• HC high conservation
• LC low conservation
• LC sequence of low conservation
• HC sequence of low conservation
• a pair of edge amino acids distinguish or separate the amino acid sequence of high conservation (HC) from the two flanking amino acid sequences of low conservation (LC) which are on either end of or otherwise bound the amino acid sequence of high concentration.
• a pair of edge amino acids distinguish or separate the amino acid sequence of low conservation (LC) from the two flanking amino acid sequences of high conservation (HC) which are on either end of or otherwise bound the amino acid sequence of low conservation.
• the middle sequence is removed to create a mutant DNA binding protein according to the methods described herein which retains DNA binding activity and which is smaller in size compared to the wild type DNA binding protein.
• the edge amino acids define the middle sequence to be removed by flanking the middle sequence or otherwise separating the middle sequence from adjacent sequences in series to create the mutant DNA binding protein.
• a middle sequence in the tandem sequences can be removed regardless of whether the middle sequence is an amino acid sequence of high conservation or an amino acid sequence of low conservation insofar as the mutant DNA binding protein retains useful DNA binding protein activity.
• a bioinformatics approach is used to identify potential domain boundaries in the Cas9 proteins.
• a multiple sequence alignment is created by re-aligning Cas9 sequences in the PFAM database (PF 13395) using MUSCLE, and the alignment is computationally conditioned for diversity and full-length sequences.
• the sequence conservation is calculated as the relative entropy of observed amino acid frequencies with respect to the average frequencies across all genes in Escherichia coli.
• a multi-scale edge filter (difference of Gaussians (DoG) band-pass filter) is applied to the conservation profile to assign potential protein domain boundaries referred to herein as conservation edges. Regions in between the conservation edges are selected for deletion in the first iteration of deletion mutants.
• DoG difference of Gaussians
• the present disclosure describes synthetic NM-Cas9 deletion mutants that are smaller in size yet retain near wild-type protein activity.
• the synthetic NM-Cas9 deletion mutants can be used to bind to DNA as a co-localization complex with guide RNA in a cell and create a double stranded break, a single stranded break or to locate an effector group near target DNA of interest to perform a desired function.
• an alignment-based domain detection method is provided to identify regions of a DNA binding protein, such as Cas9, that are dispensable for binding to DNA, and which can be removed to form a mutant DNA binding protein that is smaller in size compared to the wild type DNA binding protein. According to methods described herein, minimized Cas9 variants are generated that show robust activity in bacteria and human cells. According to aspects described herein, mutant functional DNA binding protein variants, such as mutant functional Cas9 variants, which are smaller than wild type DNA binding proteins, are provided.
• exemplary DNA binding proteins include Cas9 orthologs such as Neisseria meningitidis Cas9 (NM, GI:218767588) and Streptococcus thermophilus Cas9 (ST1, GI: 1 16627542) 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 1 10, 15644- 15649, doi: 10.1073/pnas.13135871 10 (2013) hereby incorporated by reference in its entireties.
• Cas9 orthologs such as Neisseria meningitidis Cas9 (NM, GI:218767588) and Streptococcus thermophilus Cas9 (ST1, GI: 1 16627542) which have been shown to function in both prokaryotes and higher eukaryo
• exemplary Cas9 orthologs are smaller in gene size compared to Streptococcus pyogenes Cas9 (SP, GI: 13622193), i.e. about 3200 versus 4100 base pairs.
• Aspects of the present disclosure are therefore directed to reducing the size of a Cas9 DNA binding protein so as to increase the efficiency with which the Cas9 DNA binding protein can be delivered, particularly using viral packaging technologies where gene length can greatly influence viral titer. See Kumar, M., Keller, B., Makalou, N. & Sutton, R. E. Systematic determination of the packaging limit of lentiviral vectors.
• Synthetically reducing the size of Cas9 genes allow for more complex regulatory systems and functional domains to be packaged within single vectors. According to an additional aspect, methods are provided to synthetically alter PAM specificity allowing for the generation of smaller Cas9 variants with increased targeting potential.
• methods are provided for making Cas9 chimera by exchanging the C-terminal domain of a first species of Cas9 with the C-terminal domain of a second species of Cas9.
• the present disclosure provides domain exchange 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 ST1 guideRNA and PAM specificity.
• the cell is a prokaryotic cell or a eukaryotic cell.
• the cell is a bacterial cell, a yeast cell, a plant cell or an animal cell.
• the cell is a mammalian cell.
• the RNA is between about 10 to about 500 nucleotides. According to one aspect, the RNA is between about 20 to about 100 nucleotides.
• the one or more RNAs is a guide RNA. According to one aspect, the one or more RNAs is a crRNA. According to one aspect, the one or more RNAs is a tracrRNA. According to one aspect, the one or more RNAs is a tracrRNA-crRNA fusion.
• the target DNA is genomic DNA, mitochondrial DNA, viral DNA, conjugatable element or exogenous DNA.
• the RNA guided DNA binding protein is of a Type II CRISPR
• the RNA guided DNA binding protein is a Cas9 protein that binds to the DNA and is guided by the one or more RNAs.
• Fig. 1 is an image in which E. coli cells contain a YFP reporter for NM-Cas9 activity and are transformed with various NM-Cas9 nuclease null genes.
• the cells are fluorescent (upper right quadrant-Negative control) and in the presence of full length nuclease null NM-Cas9 the cells are non- fluorescent (upper left quadrant-Positive control).
• Two of the generated NM-Cas9 deletions are shown, NM-Cas9-A255-449 shows near wild-type levels of repression (bottom left quadrant) and NM-Cas9-A874-922 shows lack of most DNA binding capacity (bottom right quadrant).
• Fig. 2 is a phylogenetic tree as described in 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 hereby incorporated by reference in its entirety. Marked in red are sequences used as an initial seed for the PFAM realignment.
• Fig. 3 is a conservation profile of Cas9 alignment after truncation to positions of NM-
• Fig. 4 is a conservation profile truncated to positions in SP-Cas9.
• Fig. 5A is a plot of first-order amino acid conservation within Cas9 proteins. Relative entropies are calculated with respect to the average amino acid frequency across all genes in Escherichia coli. Vertical lines above plot represent boundaries determined by the alignment-based boundary detection algorithm, with bold lines representing the six most significant boundaries detected.
• Fig. 5B shows 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/gktl074 (2014) hereby incorporated by reference in its entirety.
• RuvCI-III are the parts folding into the RuvC nuclease domain.
• HNH is the HNH nuclease domain.
• RRR is the arginine-rich alpha-helical region. Cross- hatched is the extension of this region based on the arginine-rich stretch around position 140.
• Fig. 6A is a schematic depicting design of NM Cas9 transcriptional reporter. The location of the protospacer and NM specific PAM are noted.
• Fig. 6C is a schematic depicting NM Cas9 domain structure. White boxes with dashed outlines give the extent of the largest excised regions from NM mutants that cause minimal alteration in DNA binding activity.
• Fig. 8B is a schematic depicting reporter construct for testing STl activation which contains a minimal CMV promoter (min CMV) upstream of a tdTomato reporter. STl nuclease null Cas9-VP64 fusion proteins binding upstream of the minimal CMV promoter lead to transcriptional activation and fluorescence within human cells.
• Fig. 8C are images of cells transfected with STl activators including deletion mutants were transfected along with sgRNAs and the tdTomato reporter and were visualized by fluorescence microscopy.
• Fig. 9 is directed to TD Cas9 deletion analysis and functional validation in E. coli.
• TD nuclease-null deletion mutants were tested using a transcriptional repressor assay.
• Fig. 10A is directed to NM-ST1 domain swap analysis as determined by a transcriptional repression assay and in particular, design of NM and STl transcriptional reporters with the sequence of the NM or STl specific PAM illustrated.
• Fig. 1 OB is a schematic depicting outline of NM and STl Cas9 with the location of the amino acid swap points noted.
• Fig. 1 1A is directed to NM-ST1 domain swap analysis as determined by a transcriptional repression assay, and in particular, design of STl transcriptional reporter with the sequence of the STl specific PAM illustrated.
• Fig. 1 1B is a schematic outline of NM and STl Cas9 with the location of the amino acid swap points noted.
• Embodiments of the present invention are directed to mutant RNA guided DNA binding proteins of the Type II CRISPR system. Such mutants are created by removing sequences that are not well conserved or are otherwise highly divergent among species within a genus of RNA guided DNA binding proteins of the Type II CRISPR system. According to one aspect, the sequences of species within a family of RNA guided DNA binding proteins are aligned and sequences of low conservation or sequences between conservation edges are determined. These sequences are then deleted from a particular RNA guided DNA binding protein. Exemplary RNA guided DNA binding proteins include Cas9 proteins present, for example, in Type II CRISPR systems. Such Cas9 proteins and Type II CRISPR systems are well documented in the art.
• mutant DNA binding proteins described herein can be used to make double stranded cuts in target DNA, single stranded cuts in target DNA or to bind to target DNA in a manner to locate an effector group near the target DNA such that that effector group can interact with the target DNA.
• effector groups include activators, repressors or epigenetic modifiers known to those of skill in the art.
• Exemplary DNA binding proteins having nuclease activity function to nick or cut double stranded DNA. Such nuclease activity may result from the DNA binding protein having one or more polypeptide sequences exhibiting nuclease activity. Such exemplary DNA binding proteins may have two separate nuclease domains with each domain responsible for cutting or nicking a particular strand of the double stranded DNA.
• Exemplary polypeptide sequences having nuclease activity known to those of skill in the art include the McrA-HNH nuclease related domain and the RuvC-like nuclease domain. Accordingly, exemplary DNA binding proteins are those that in nature contain one or more of the McrA-HNH nuclease related domain and the RuvC-like nuclease domain.
• a DNA binding protein having two or more nuclease domains may be modified or altered to inactivate all but one of the nuclease domains.
• a DNA binding protein nickase Such a modified or altered DNA binding protein is referred to as a DNA binding protein nickase, to the extent that the DNA binding protein cuts or nicks only one strand of double stranded DNA.
• the DNA binding protein nickase is referred to as an RNA guided DNA binding protein nickase.
• An exemplary DNA binding protein is an RNA guided DNA binding protein of a Type II CRISPR System.
• An exemplary DNA binding protein is a Cas9 protein.
• Cas9 generates a blunt-ended double-stranded break 3bp upstream of the protospacer-adjacent motif (PAM) via a process mediated by two catalytic domains in the protein: an HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that cleaves the non-complementary strand.
• PAM protospacer-adjacent motif
• Cas9 proteins are known to exist in many Type II CRISPR systems including the following as identified in the supplementary information to Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 201 1, pp. 467-477: Methanococcus maripaludis C7; Corynebacterium diphtheriae; Corynebacterium efficiens YS-314; Corynebacterium glutamicum ATCC 13032 Kitasato; Corynebacterium glutamicum ATCC 13032 Bielefeld; Corynebacterium glutamicum R; Corynebacterium kroppenstedtii DSM 44385; Mycobacterium abscessus ATCC 19977; Nocardia farcinica IFM10152; Rhodococcus erythropolis PR4; Rhodococcus jostii RHA1; Rhodococcus opacus B4 uid36573; Acidothermus cellulolyticus 1 1B; Arthrobacter chlorophenolicus A
• aspects of the present disclosure are directed to a mutant of a Cas9 protein present in a Type II CRISPR system, such as any one of the species identified above.
• An exemplary Cas9 protein is that found in Neisseria meningitides, such as Neisseria meningitides 053442; Neisseria meningitides alphal4; Neisseria meningitides Z2491.
• Cells according to the present disclosure include any cell into which foreign nucleic acids can be introduced and expressed as described herein. It is to be understood that the basic concepts of the present disclosure described herein are not limited by cell type.
• Cells according to the present disclosure include eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, archael cells, eubacterial cells and the like.
• 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.
• Target nucleic acids include any nucleic acid sequence to which a mutant RNA guided DNA binding protein nuclease can be useful to nick or cut.
• Target nucleic acids include genes.
• DNA such as double stranded DNA
• a co-localization complex can bind to or otherwise co-localize with the DNA at or adjacent or near the target nucleic acid and in a manner in which the co-localization complex may have a desired effect on the target nucleic acid.
• target nucleic acids can include endogenous (or naturally occurring) nucleic acids and exogenous (or foreign) nucleic acids.
• DNA includes genomic DNA, mitochondrial DNA, viral DNA, a conjugatable element or exogenous DNA.
• Foreign nucleic acids i.e. those which are not part of a cell's natural nucleic acid composition
• Methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like.
• transfection transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like.
• the genetic material required to encode a Cas9 protein is reduced by deleting portions of the Cas9 protein which are not well conserved or otherwise diverge within species within a family of Cas9 or are between conservation edges within species within a family of Cas9.
• additional nucleic acids can be included with a vector designed to deliver the Cas9, such as nucleic acids encoding guide R A or regulatory elements or effector domains. If one uses the smallest characterized Cas9 family member, -4,500 kilobases of DNA will be required to encode for the necessary genetic elements (Cas9 protein and gRNA) in order to properly localize Cas9 to the desired genomic locus.
• Cas9 is near the size limit for packaging within AAV based viral vector (which is a regulatory approved viral vector in Europe.) Further, some of the first transcriptional and epigenetic effector domains to be fused to programmable DNA binding proteins are greater than 2,000 basepairs and thus far out of the packaging limit for AAV vectors and approaching the limit of lentiviral packaging systems (-8,000 basepairs) once fused to Cas9.
• NM-Cas9 Neisseria meningitides Cas9
• NM-Cas9 3249 bp in size. Requirements for targeting to the genome and the residues involved in nuclease activity are determined.
• an alignment of Cas9 proteins was generated and contiguous stretches of low conservation or stretches between conservation edges were identified for deletion. Several regions of interest were identified and selectively removed from NM-Cas9 which was then assessed for function by using a Cas9 repressor assay.
• NM-Cas9 In the assay, a variant of NM-Cas9 was used that lacks nuclease activity but is able to be targeted to the 5' region of a reporter gene. If NM-Cas9 is able to bind to the reporter gene it will repress transcription and in the case of a fluorescent reporter, the cells will appear non- fluorescent.
• Cas9 alignment and deletion prediction Full length sequences of Cas9 homologs were obtained either from the PFAM database or from a database search such as jackHMMER (R.D. Finn, J. Clements, S.R. Eddy, Nucleic Acids Research (201 1) Web Server Issue 39:W29-W37 hereby incorporated by reference in its entirety).
• an alignment is 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 (201 1) hereby incorporated by reference in its entirety), or equivalent.
• 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 (201 1) hereby incorporated by reference in its entirety
• the alignment was computationally cut to the positions of the sequence of interest and conditioned to diminish alignment bias (e.g. sequences with a greater than 95% pairwise identity were removed).
• Conservation is calculated as the entropy or relative entropy of amino acid frequencies per position, taking into account the amount of amino acids and gaps at that position. Deletions are targeted towards regions of low conservation or between conservation edges. In
• cells are co-transformed with synthetic NM-Cas9 variants and the reporter plasmid.
• the doubly transformed cells are then grown up at 37°C, and the amount of YFP fluorescence is measured using a fluorescence plate reader and compared to cells that are transformed with a control plasmid with wild-type nuclease null NM- Cas9 and the reporter plasmid.
• mutant Cas9 proteins which have 1000 fewer base pairs or 900 fewer base pairs compared to the wild type Cas9, such as NM-Cas9 and retain near wild-type levels of activity.
• a functional NM-Cas9 allele lacking the HNH motif and surrounding nucleotides NM-Cas9-A567-654 was made which retained near wild-type ability to bind DNA as determined by the YFP reporter assay.
• aspects of the present disclosure include a high-throughput approach for random deletion creation and screening of functional mutants.
• plasmid DNA containing the desired Cas9 allele can be sheared using a promiscuous nuclease, sonication, repeatedly pipetting the sample, or other chemical, enzymatic or environmental means. Once fragmented, the plasmid DNA can be treated with exonucleases to remove nucleotides from the Cas9 gene.
• fragmented ends are made blunt ended with enzymes such as Mung Bean nuclease or Klenow polymerase and ligated together to regenerate a Cas9 plasmid containing a random deletion.
• enzymes such as Mung Bean nuclease or Klenow polymerase
• ligated together to regenerate a Cas9 plasmid containing a random deletion.
• an exogenous domain such as a linker or effector motif within the deleted portion of Cas9
• Such domains can be ligated to the blunt ended fragmented DNA, and subsequent circularization of the plasmid will produce a Cas9 coding sequence where the exogenous domain has been inserted within the deleted portion of Cas9.
• the library of circularized molecules will then be transformed into E. coli and plasmid DNA will be extracted.
• the library can be transformed into cells containing a reporter assay for Cas9 activity and members of the library that maintain functional activity can be identified.
• the coding sequence for Cas9 from the newly generated library can be isolated via digestion or PCR and the fragments can be size-selected to be shorter than the initial wild-type Cas9 gene. These smaller members can then be ligated back into the starting vector and transformed into cells containing the reporter of Cas9 activity.
• a library of oligonucleotides can be generated that have 3 ' homology to the Cas9 gene but contain 5' homology to each other, where the 3' end of each oligonucleotide binds to a different stretch of around 30 basepairs within Cas9. These oligonucleotides cover both the sense and anti-sense strands of the Cas9 coding sequence. PCR can then be performed with these oligonucleotides to generate a series of Cas9 fragments with each product from a given sense PCR reaction having complementarity to all other anti-sense PCR products and vice-versa.
• fragments can then be annealed together using methods such as Gibson assembly or overlap extension PCR followed by ligation into a vector backbone and transformed into cells, generating a library of Cas9 variants with random stretches of the Cas9 gene removed.
• the oligonucleotides on their 5' ends should contain complementarity towards the longer linker or effector domain and this domain should then be included in the Gibson assembly reaction or during overlap extension PCR.
• Cas9 nuclease null plasmids were STl (Addgene# 48659) or were constructed from plasmids NM and TD (Addgene# 48646 and 48648, respectively) by introducing the following point mutations (NM: D16A D587A H588A N611A and TD: D13A D878A H879A N902A).
• Cas9 deletions were generated using Gibson assembly. Internal deletions when made were joined by a 5 amino acid Ser-Gly-Gly-Gly-Ser linker, except for NM ⁇ 566-620 which lacks a linker between joined fragments.
• the N-terminal domain exchange fused residues 1- 117 of STl onto residues 1 18-1082 of NM.
• the C-terminal domain exchange fused residues 1-727 of NM onto residues 743- 1 121 of ST1.
• Reporter constructs used for analysis of the deletion mutants are similar to those previously published except they combine the spacer element and YFP reporter into a single SClOl-kanR plasmid backbone. Reporter constructs for domain-exchange analysis are identical to those used previously. See Esvelt, K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature methods 10, 1 1 16-1 121, doi: 10.1038/nmeth.2681 (2013) hereby incorporated by reference in its entirety.
• Cas9 repression assays were performed by co-transforming NEB 10-beta cells (New England BioLabs) with the appropriate spacer/reporter construct and Cas9 vector to be investigated. Colonies from transformations were picked and grown at 37°C with continuous shaking in 96 well plates. Plates were read the following day using a Synergy Neo microplate reader (BioTek), measuring fluorescence at 495-528 nm and absorbance at 600 nm.
• a and B Two different previously published spacer/protospacer combinations (A and B) (see Esvelt, K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature methods 10, 1 1 16-1 121, doi: 10.1038/nmeth.2681 (2013)) were tested. For all other experiments, only spacer/protospacer combination B was examined.
• Example VIII Example VIII
• HEK 293T cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with high glucose supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin (Invitrogen). Cells were maintained at 37°C and 5% C0 2 in a humidified incubator. Cells were transfected in 24 well plates seeded with 50,000 cells per well. 400ng of Cas9 activator, lOOng of gRNA and 60ng of reporter plasmid were delivered to each well using 2.5 ul of Lipofectamine 2000. Cells were grown an additional 36-48 hours before being assays using immunofluorescence or FACS.
• sequences were then split into two groups, one with and one without the large insertion at approximately position 150 (which distinguishes e.g. between NM-Cas9 and ST-Cas9 on one hand and SP-Cas9 and TD-Cas9 on the other).
• These groups are separately aligned using MUSCLE (see Edgar, RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Res 32, 1792-97, re-aligned using a windowed approach (because of the length of the sequences), and then profile-profile aligned back into one seed alignment.
• the sequences in PF 13395 are realigned with MUSCLE and using the seed alignment. All alignments using a seed are performed by aligning each of the target sequences one-by-one to the seed. This alignment is used to determine the top-hit identity between seed and target sequences, which are re-ordered according to decreasing top-hit identity. The target sequences are then again aligned to the seed one -by-one, now in order of decreasing identity. This two-step approach is taken to ensure the robustness of the alignment. Also these sequences are split depending on whether they contain the insertion or not, and the two separate groups are re-aligned with the seed as a profile. Short sequences and sequences with large truncations are removed manually. Sequences with higher than 90% pairwise similarity are removed.
• Sequence conservation was calculated as the relative entropy with respect to the background frequency of amino acids averaged over all genes in Escherichia coli 0157. 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) hereby incorporated by reference in its entirety) to the resulting conservation profile, averaging over multiple length scales to achieve robustness to choice of parameters and detection at various length scales.
• DoG Difference of Gaussians
• the filter correctly identifies the known HNH and RuvC domain arrangements that were assigned previously in Sapranauskas, R. et al.
• the Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.
• Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.
• NM positions 254-449 represent a stretch of relatively low conservation, in a region of the protein that is specific to Cas9 proteins.
• Positions 567 to 654 represent the HNH domain, a domain known to be critical in Cas9 DNA catalysis but was found to be dispensable for DNA binding.
• NM-Cas9 regions removed from NM-Cas9 were not unique to NM but represent general regions that can be removed from other Cas9 family members
• the corresponding deletions were generated within nuclease null-variants of Streptococcus thermophilus Cas9 (ST1) and Treponema denticola Cas9 (TD, GL42525843) and their function with the transcriptional repression assay was measured (Fig. 8A and Fig. 9).
• ST1 and TD results similar to their wild-type counterparts, suggesting that the removed regions are dispensable for Cas9 DNA binding throughout the Cas9 phylogeny, even among more distant members within the type II-A subfamily such as TD.
• the Cas9 N- and C-terminal domains may play critical roles in crRNA:tracrR A binding and/or PAM selectivity.
• a series of domain exchange mutants between NM and STl were made, replacing the N and/or C terminus of NM with the homologous region from STl .
• the chimeric proteins were then tested using the transcriptional reporter assay described herein altering the guideRNA and/or Cas9 specific PAM within the reporter to determine the influence of the domain exchanges on protein specificity (Fig. 10A).
• the exact positions for the domain swaps were determined based on domain boundary analysis: positions were selected that were as close as possible to the most significant N- and C-terminal boundaries identified (Fig.

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