Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing
AssigneeThe General Hospital Corporation
InventorsJ. Keith Joung, Jeffry D. Sander, Yan-fang Fu, Morgan Maeder
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Abstract
CRISPR-Cas genome editing uses a guide RNA, which includes both a complementarity region, which binds the target DNA by base-pairing, and a Cas9-binding region, to direct a Cas9 nuclease to a target DNA. Further disclosed are methods for increasing specificity of RNA-guided genome editing using CRISPR/Cas9 systems by using truncated guide RNAs (tru-gRNAs).
Claims11
A method of increasing specificity of S. pyogenes CRISPR-Cas9 (Cas9) RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA that includes a complementarity region at the 5′ end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, wherein the selected target genomic sequence is immediately 5′ of a protospacer adjacent motif (PAM), the guide RNA comprises
- SEQ ID NO:4, and wherein in the presence of a S. pyogenes Cas9 genome editing enzyme, the guide RNA complementarity region binds and directs the Cas9 genome editing enzyme to the selected target genomic sequence, thereby increasing specificity of RNA-guided genome editing in a cell.
A method of inducing a break in a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell:
- a S. pyogenes CRISPR/Cas9 nuclease or nickase;
- and
- a guide RNA that includes a complementarity region at the 5′end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a double-stranded DNA molecule comprising a target sequence, wherein the target sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA complementarity region binds and directs the Cas9 nuclease or nickase to the target region of a double-stranded DNA molecule, and wherein the guide RNA comprises
- SEQ ID NO:4, thereby inducing a break in the target region of a double-stranded DNA molecule in a cell.
A method of modifying a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell:
- a S. pyogenes CRISPR dCas9-heterologous functional domain fusion protein (dCas9-HFD);
- and
- a guide RNA that includes a complementarity region at the 5′end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target sequence present on a double-stranded DNA molecule, wherein the selected target sequence is immediately 5′ of a protospacer adjacent motif (PAM), the guide RNA comprises
- SEQ ID NO:4, and wherein the guide RNA complementarity region binds and directs the dCas9-HFD to the selected target sequence, thereby modifying a target region of a double-stranded DNA molecule in a cell.
Dependencies are inferred from the claim text.