Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing
AssigneeThe General Hospital Corporation
InventorsJ. Keith Joung, Jeffry D. Sander, Morgan Maeder, Yanfang Fu
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Abstract
Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).
Claims17
A method of increasing specificity of Streptococcus 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 target sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA is
- (i) a single guide RNA that includes at the 5′ end of the single guide RNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of the selected target genomic sequence on a double-stranded DNA molecule, or
- (ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of the selected target genomic sequence, and a tracrRNA,
- 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 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, wherein the target region 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 sequence of a double-stranded DNA molecule, and wherein the guide RNA is
- (i) a single guide RNA that includes at the 5′ end of the single guide RNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence on a double stranded DNA molecule, or
- (ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA;
- 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 target sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA is:
- (i) a single guide RNA that includes a complementarity region at the 5′ end of the single 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 on a double stranded DNA molecule, or
- (ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA, and
- wherein the guide RNA complementarity region binds and directs the dCas9-HFD to the target region of the double-stranded DNA molecule, thereby modifying the target region of a double-stranded DNA molecule in a cell.
Dependencies are inferred from the claim text.