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US 10,479,982 B2Granted
Engineered CRISPR-Cas9 nucleases with altered PAM specificity
AssigneeThe General Hospital Corporation
InventorsJ. Keith Joung, Benjamin Kleinstiver
Life of this patent
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Abstract
Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.
Claims35
An isolated variant Streptococcus pyogenes Cas9 (SpCas9) protein, comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:
- 1, with a mutation at G1218 and optionally at one or more of the following positions:
- G1104, S1109, L1111, E1219, D1135, N1317, R1335, T1337, wherein the isolated variant SpCas9 protein can interact with a guide RNA and a target DNA.
A method of altering the genome of a cell, the method comprising expressing in the cell, or contacting the cell with,
- (i) an isolated variant Streptococcus pyogenes Cas9 (SpCas9) protein, comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:
- 1, with a mutation at G1218 and optionally at one or more of the following positions:
- G1104, S1109, L1111, E1219, D1135, N1317, R1335, T1337, wherein the isolated variant SpCas9 protein can interact with a guide RNA and a target DNA, or
- (ii) a fusion protein comprising an isolated variant Streptococcus pyogenes Cas9 (SpCas9) protein fused to a heterologous functional domain, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein, wherein the isolated variant SpCas9 protein comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:
- 1, with a mutation at G1218 and optionally at one or more of the following positions:
- G1104, S1109, L1111, E1219, D1135, N1317, R1335, T1337, and wherein the isolated variant SpCas9 protein can interact with a guide RNA and a target DNA, and
- a guide RNA having a region complementary to a selected portion of the genome of the cell.
4 more elements
A method of altering a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with
- (i) an isolated variant Streptococcus pyogenes Cas9 (SpCas9) protein, comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:
- 1, with a mutation at G1218 and optionally at one or more of the following positions:
- G1104, S1109, L1111, E1219, D1135, N1317, R1335, T1337, wherein the isolated variant SpCas9 protein can interact with a guide RNA and a target DNA, or
- (ii) a fusion protein comprising an isolated variant Streptococcus pyogenes Cas9 (SpCas9) protein fused to a heterologous functional domain, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein, wherein the isolated variant SpCas9 protein comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:
- 1, with a mutation at G1218 and optionally at one or more of the following positions:
- G1104, S1109, L1111, E1219, D1135, N1317, R1335, T1337, and wherein the isolated variant SpCas9 protein can interact with a guide RNA and a target DNA,
- and a guide RNA having a region complementary to a selected portion of the dsDNA molecule.
4 more elements
Dependencies are inferred from the claim text.