# US 2016/0257973 A1 · Methods for increasing cas9-mediated engineering efficiency > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2016/0257973 A1 - Document ID: US20160257973A1 - Status: Published application - Kind: A1 - Application number: 14/836,753 - Filed: 2015-08-26 - Published: 2016-09-08 - Earliest claimed: 2014-08-27 - Assignees: Caribou Biosciences, Inc. - Applicants: Caribou Biosciences, Inc. - Inventors: Peter Sean Cameron; Rachel E. Haurwitz; Andrew P. May; Christopher H. Nye; Megan van Overbeek - Web page: https://patentgrep.com/patent/US20160257973A1 ## CPC - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 15/907: {in mammalian cells} - C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein} - C12N 15/102: {Mutagenizing nucleic acids} - C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids} - C12Y 301/30: Endoribonucleases active with either ribo- or deoxyribonucleic acids and producing 5'-phosphomonoesters (3.1.30) - C12N 2310/10: Type of nucleic acid - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract Methods for use with Type II CRISPR-Cas9 systems for increasing Cas9-mediated genome engineering efficiency are disclosed. The methods can be used to decrease the number of off-target nucleic acid double-stranded breaks and/or to enhance homology-directed repair of a cleaved target nucleic acid. ## Claims ### Claim 1 A method for reducing binding and/or cleavage of an off-target nucleic acid by a complex comprising a catalytically active Cas9 protein and a guide polynucleotide, said method comprising: (a) contacting a first complex with a selected target nucleic acid, wherein said first complex comprises: (i) a catalytically active Cas9 protein and (ii) a first guide polynucleotide that comprises a spacer adapted to bind to said selected target nucleic acid; and (b) contacting a second complex with an off-target nucleic acid, wherein said second complex comprises (i) a catalytically inactive Cas9 protein (dCas9 protein) that does not cleave the off-target nucleic acid and (ii) a second guide polynucleotide that comprises a spacer adapted to bind to said off-target nucleic acid, thereby reducing binding and/or cleavage by said first complex of said off-target nucleic acid. ### Claim 2 (depends on 1) The method of claim 1, wherein the guide polynucleotide is a single-guide RNA (sgRNA). ### Claim 3 (depends on 1) The method of claim 1, wherein said catalytically active Cas9 protein comprises at least 75% amino acid identity to a Cas9 from Streptococcus pyogenes, with the proviso that said Cas9 protein retains catalytic activity. ### Claim 4 (depends on 1) The method of claim 1, wherein said catalytically active Cas9 protein comprises at least 95% amino acid identity to a Cas9 from S. pyogenes, with the proviso that said Cas9 protein retains catalytic activity. ### Claim 5 (depends on 1) The method of claim 1, wherein said catalytically active Cas9 protein is a S. pyogenes Cas9 protein or an orthologous Cas9 protein. ### Claim 6 (depends on 1) The method of claim 1, wherein said dCas9 protein comprises at least one mutation in one or more endonuclease domains to render the dCas9 protein catalytically inactive. ### Claim 7 (depends on 6) The method of claim 6, wherein said dCas9 protein comprises at least 75% amino acid identity to a Cas9 protein from S. pyogenes. ### Claim 8 (depends on 6) The method of claim 6, wherein said dCas9 protein comprises at least 95% amino acid identity to a Cas9 protein from S. pyogenes. ### Claim 9 (depends on 6) The method of claim 6, wherein said dCas9 protein is a S. pyogenes Cas9 protein, or an orthologous Cas9 protein with at least one mutation in one or more endonuclease domains to render the orthologous Cas9 protein catalytically inactive. ### Claim 10 (depends on 6) The method of claim 6, wherein said one or more mutations is in a RuvC-1 domain. ### Claim 11 (depends on 6) The method of claim 6, wherein said one or more mutations is in a HNH domain. ### Claim 12 (depends on 6) The method of claim 6, wherein said mutations are in both RuvC-1 and HNH domains. ### Claim 13 (depends on 10) The method of claim 10, wherein said one or more mutations comprises a D10A mutation, numbered relative to S. pyogenes Cas9, or the corresponding mutation in an orthologous Cas9 protein. ### Claim 14 (depends on 11) The method of claim 11, wherein said one or more mutations comprises a H840A mutation, numbered relative to S. pyogenes Cas9, or the corresponding mutation in an orthologous Cas9 protein. ### Claim 15 (depends on 6) The method of claim 6, wherein said one or more mutations comprises a D10A mutation and a H840A mutation, numbered relative to S. pyogenes Cas9, or the corresponding mutations in an orthologous Cas9 protein. ### Claim 16 (depends on 1) The method of claim 1, wherein said binding and/or cleavage of said first complex to said off-target nucleic acid is reduced by at least 30%. ### Claim 17 (depends on 1) The method of claim 1, wherein said binding and/or cleavage of said first complex to said off-target nucleic acid is reduced by at least 70%. ### Claim 18 (depends on 1) The method of claim 1, wherein said selected target nucleic acid is DNA. ### Claim 19 (depends on 18) The method of claim 18, wherein said selected target nucleic acid is double-stranded DNA. ### Claim 20 (depends on 1) The method of claim 1, wherein the selected target nucleic acid is cleaved to provide a cleavage site, and said method further comprises modifying said target nucleic acid. ### Claim 21 (depends on 20) The method of claim 20, wherein said modifying comprises inserting at least a portion of a donor polynucleotide at said cleavage site. ### Claim 22 (depends on 20) The method of claim 20, wherein said modifying comprises deleting one or more nucleotides at said cleavage site. ### Claim 23 (depends on 1) The method of claim 1, wherein said method is performed in a cell. ### Claim 24 (depends on 23) The method of claim 23, wherein said cell is a eukaryotic cell. ### Claim 25 (depends on 1) The method of claim 1, wherein said method is performed in vitro. ### Claim 26 A method for cleaving a single strand of a target nucleic acid comprising contacting first and second complexes at spaced-apart locations on the same strand of a nucleic acid molecule, wherein: (a) said first complex comprises (i) a first Cas9 protein with a mutation in an endonuclease domain thereof to render the Cas9 protein a nickase; and (ii) a first guide polynucleotide that comprises a spacer adapted to bind to a first target nucleic acid; and (b) said second complex comprises (i) a second Cas9 protein with a mutation in an endonuclease domain thereof, to render the Cas9 protein a nickase; and (ii) a second guide polynucleotide that comprises a spacer adapted to bind to a second target nucleic; wherein said first and second Cas9 proteins cleave a single strand of said nucleic acid molecule at the spaced-apart locations on the same strand, to render a single-stranded break. ### Claim 27 (depends on 26) The method of claim 26, wherein the guide polynucleotide is a single-guide RNA (sgRNA). ### Claim 28 (depends on 26) The method of claim 26, wherein said first Cas9 protein and/or said second Cas9 protein comprises at least 75% amino acid identity to a Cas9 protein from S. pyogenes. ### Claim 29 (depends on 28) The method of claim 28, wherein said first Cas9 protein and/or said second Cas9 protein comprises at least 95% amino acid identity to a Cas9 protein from S. pyogenes. ### Claim 30 (depends on 26) The method of claim 26, wherein said first Cas9 protein and/or said second Cas9 protein is a S. pyogenes Cas9 protein, or an orthologous Cas9 protein with a mutation in an endonuclease domain thereof, to render the orthologous Cas9 protein a nickase. ### Claim 31 (depends on 26) The method of claim 26, wherein said mutation in said first Cas9 protein and/or said second Cas9 protein is in a RuvC-1 domain. ### Claim 32 (depends on 26) The method of claim 26, wherein said mutation in said first Cas9 protein and/or said second Cas9 protein is in a HNH domain. ### Claim 33 (depends on 31) The method of claim 31, wherein said mutation in said first Cas9 protein and/or said second Cas9 protein comprises a D10A mutation, numbered relative to S. pyogenes Cas9, or the corresponding mutation in an orthologous Cas9 protein. ### Claim 34 (depends on 32) The method of claim 32, wherein said mutation in said first Cas9 protein and/or said second Cas9 protein comprises a H840A mutation, numbered relative to S. pyogenes Cas9, or the corresponding mutation in an orthologous Cas9 protein. ### Claim 35 (depends on 26) The method of claim 26, wherein said target nucleic acid is double-stranded DNA. ### Claim 36 (depends on 35) The method of claim 35, wherein said first and second complexes bind to and cleave the anti-sense strand of said double-stranded DNA. ### Claim 37 (depends on 35) The method of claim 35, wherein said first and second complexes bind to and cleave the sense strand of said double-stranded DNA. ### Claim 38 (depends on 26) The method of claim 26, wherein said method further comprises modifying said target nucleic acid by inserting at least a portion of a donor polynucleotide into said target nucleic acid at the single-stranded break. ### Claim 39 (depends on 38) The method of claim 38, wherein said donor polynucleotide is single-stranded. ### Claim 40 (depends on 38) The method of claim 38, wherein said inserting is done using homology-directed repair of the donor polynucleotide with the target nucleic acid. ### Claim 41 (depends on 26) The method of claim 26, wherein said method is performed in a cell. ### Claim 42 (depends on 41) The method of claim 41, wherein said cell is a eukaryotic cell. ### Claim 43 (depends on 26) The method of claim 26, wherein said method is performed in vitro. ### Claim 44 A method for directing homology-directed repair (HDR) in a target nucleic acid comprising: (a) contacting a first complex with a first target nucleic acid, wherein said first complex comprises: (i) a catalytically active Cas9 protein and (ii) a first guide polynucleotide that comprises a spacer adapted to bind to said first target nucleic acid, wherein said first complex cleaves the first target nucleic acid; and (b) contacting a second complex with a second target nucleic acid, wherein said second complex comprises: (i) a first catalytically inactive Cas9 protein (dCas9 protein) that comprises at least one mutation in one or more endonuclease domains to render the dCas9 protein catalytically inactive such that the dCas9 protein does not cleave the second target nucleic acid, and (ii) a second guide polynucleotide that comprises a spacer adapted to bind to said second target nucleic acid, wherein the second complex comprises one end of a polynucleotide donor associated therewith and configured in proximity to the cleaved first target nucleic acid; wherein at least a portion of the polynucleotide donor is inserted into the first target nucleic acid via HDR. ### Claim 45 (depends on 44) The method of claim 44, wherein the first and second guide polynucleotides are sgRNAs. ### Claim 46 (depends on 44) The method of claim 44, wherein the second target nucleic acid is upstream of the first target nucleic acid. ### Claim 47 (depends on 44) The method of claim 44, wherein the second target nucleic acid is downstream of the first target nucleic acid. ### Claim 48 (depends on 44) The method of claim 44, further comprising: (c) contacting a third complex with a third target nucleic acid, wherein the third target nucleic acid is positioned downstream of the first target nucleic acid when the first target nucleic acid is downstream of the second target nucleic acid, or wherein the third target nucleic acid is positioned upstream of the first target nucleic acid when the first target nucleic acid is upstream of the second target nucleic acid, wherein said third complex comprises: (i) a second dCas9 protein that comprises at least one mutation in one or more endonuclease domains to render the second dCas9 protein catalytically inactive such that the second dCas9 protein does not cleave the third target nucleic acid, and (ii) a third guide polynucleotide that comprises a spacer adapted to bind to said third target nucleic acid, and wherein the third complex comprises the other end of the polynucleotide donor associated with the second complex. ### Claim 49 (depends on 48) The method of claim 48, wherein the guide polynucleotide is a sgRNA. ### Claim 50 (depends on 44) The method of claim 44, wherein the 5′ end of the polynucleotide donor is associated with the second complex. ### Claim 51 (depends on 44) The method of claim 44, wherein the 3′ end of the polynucleotide donor is associated with the second complex. ### Claim 52 (depends on 48) The method of claim 48, wherein the 5′ end of the polynucleotide donor is associated with the second complex and the 3′ end of the polynucleotide donor is associated with the third complex. ### Claim 53 (depends on 48) The method of claim 48, wherein the 3′ end of the polynucleotide donor is associated with the second complex and the 5′ end of the polynucleotide donor is associated with the third complex. ### Claim 54 (depends on 44) The method of claim 44, wherein said catalytically active Cas9 protein comprises at least 75% amino acid identity to a Cas9 protein from S. pyogenes, with the proviso that said Cas9 protein retains catalytic activity. ### Claim 55 (depends on 44) The method of claim 44, wherein said catalytically active Cas9 protein comprises at least 95% amino acid identity to a Cas9 protein from S. pyogenes, with the proviso that said Cas9 protein retains catalytic activity. ### Claim 56 (depends on 44) The method of claim 44, wherein said catalytically active Cas9 protein is a S. pyogenes Cas9 protein or an orthologous Cas9 protein. ### Claim 57 (depends on 44) The method of claim 44, wherein said dCas9 protein comprises at least 75% amino acid identity to a Cas9 protein from S. pyogenes. ### Claim 58 (depends on 44) The method of claim 44, wherein said dCas9 protein comprises at least 95% amino acid identity to a Cas9 protein from S. pyogenes. ### Claim 59 (depends on 44) The method of claim 44, wherein said dCas9 protein is a S. pyogenes Cas9 protein, or an orthologous Cas9 protein with at least one mutation in one or more endonuclease domains to render the S. pyogenes Cas9 protein or the orthologous Cas9 protein catalyitcally inactive. ### Claim 60 (depends on 44) The method of claim 44, wherein said one or more mutations is in a RuvC-1 domain. ### Claim 61 (depends on 44) The method of claim 44, wherein said one or more mutations is in a HNH domain. ### Claim 62 (depends on 60) The method of claim 60, wherein said one or more mutations comprises a D10A mutation, numbered relative to S. pyogenes Cas9, or the corresponding mutation in an orthologous Cas9 protein. ### Claim 63 (depends on 61) The method of claim 61, wherein said one or more mutations comprises a H840A mutation, numbered relative to S. pyogenes Cas9 or the corresponding mutation in an orthologous Cas9 protein. ### Claim 64 (depends on 44) The method of claim 44, wherein said one or more mutations comprises a D10A mutation and a H840A mutation, numbered relative to S. pyogenes Cas9, or the corresponding mutations in an orthologous Cas9 protein. ### Claim 65 (depends on 44) The method of claim 44, wherein said target nucleic acids are DNA. ### Claim 66 (depends on 44) The method of claim 44, wherein said method is performed in a cell. ### Claim 67 (depends on 66) The method of claim 66, wherein said cell is a eukaryotic cell. ### Claim 68 (depends on 44) The method of claim 44, wherein said method is performed in vitro.