Systems and Methods for Genomic Editing
AssigneeIntellia Therapeutics, Inc.
InventorsSabin Mulepati, Christian Dombrowski, Jennifer Lauren Curran
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Abstract
Methods and compositions for genetically modifying a cell are provided.
Claims70
A method of producing a modification in the genome of a target cell, the method comprising contacting the cell with:
- (a) a fusion protein, or a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a first cleavase and a second cleavase, wherein:
- a. the first cleavase is a Streptococcus pyogenes (Spy)Cas9 cleavase, said SpyCas9 cleavase comprising a R1333K mutation within its protospacer adjacent motif recognition domain;
- and
- b. the second cleavase is a Neisseria meningitidis (Nme)Cas9 cleavase, a Campylobacter jejuni (Cje) Cas9 cleavase, or a Simonsiella muelleri (Smu) Cas9 cleavase;
- and
- (b) a first guide RNA that directs the first cleavase to a first genomic locus;
- and
- (c) a second guide RNA that directs the second cleavase to a second genomic locus,
- wherein the second genomic locus is different from the first genomic locus.
A method of producing a cell or a population of cells comprising a modification in the genome of the target cell or cells, the method comprising contacting the cell or cells with:
- (a) a fusion protein, or a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a first cleavase and a second cleavase, wherein:
- a. the first cleavase is a S. pyogenes (Spy)Cas9 cleavase, said SpyCas9 cleavase comprising a R1333K mutation within its protospacer adjacent motif recognition domain;
- and
- b. the second cleavase is a N. meningitidis (Nme)Cas9 cleavase, a C. jejuni (Cje)Cas9 cleavase, or a S. muelleri (Smu)Cas9 cleavase;
- and
- (b) a first guide RNA that directs the first cleavase to a first genomic locus;
- and
- (c) a second guide RNA that directs the second cleavase to a second genomic locus,
- wherein the second genomic locus is different from the first genomic locus.
The method of any one of the preceding claims, wherein the first guide RNA is a single guide RNA (sgRNA), optionally a SpyCas9 guide RNA.
A polynucleotide comprising an open reading frame (ORF) encoding a fusion protein, wherein the fusion protein comprises a first cleavase and a second cleavase, wherein:
- a. the first cleavase is a S. pyogenes (Spy)Cas9 cleavase, said SpyCas9 cleavase comprising a R1333K mutation within its protospacer adjacent motif recognition domain;
- and
- b. the second cleavase is a N. meningitidis (Nme)Cas9 cleavase, a C. jejuni (Cje) Cas9 cleavase, or a S. muelleri (Smu) Cas9 cleavase.
A composition comprising
- (a) a polynucleotide comprising an open reading frame (ORF) encoding a fusion protein, wherein the fusion protein comprises a first cleavase and a second cleavase, wherein:
- a. the first cleavase is a S. pyogenes (Spy)Cas9 cleavase, said SpyCas9 cleavase comprising a R1333K mutation within its protospacer adjacent motif recognition domain;
- and
- b. the second cleavase is a N. meningitidis (Nme)Cas9 cleavase, a C. jejuni (Cje) Cas9 cleavase, or a Simonsiella muelleri (Smu) Cas9 cleavase;
- and
- (b) a first guide RNA that directs the first cleavase to a first genomic locus;
- and
- (c) a second guide RNA that directs the second cleavase to a second genomic locus,
- wherein the second genomic locus is different from the first genomic locus.
One or more lipid nanoparticles comprising:
- (a) a polynucleotide comprising an open reading frame (ORF) encoding a fusion protein, wherein the fusion protein comprises a first cleavase and a second cleavase, wherein:
- a. the first cleavase is a S. pyogenes (Spy)Cas9 cleavase, said SpyCas9 cleavase comprising a R1333K mutation within its protospacer adjacent motif recognition domain;
- and
- b. the second cleavase is a N. meningitidis (Nme)Cas9 cleavase, a C. jejuni (Cje) Cas9 cleavase, or a Simonsiella muelleri (Smu) Cas9 cleavase;
- and
- (b) a first guide RNA that directs the first cleavase to a first genomic locus;
- and
- (c) a second guide RNA that directs the second cleavase to a second genomic locus,
- wherein the second genomic locus is different from the first genomic locus.
Dependencies are inferred from the claim text.