SYSTEMS AND METHODS FOR ONE-SHOT GUIDE RNA (ogRNA) TARGETING OF ENDOGENOUS AND SOURCE DNA
AssigneeEditas Medicine, Inc.
InventorsAri E. Friedland, Hariharan Jayaram, Barrett Ethan Steinberg
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Abstract
Engineered nucleic acids encoding genome editing system components are provided, as are engineered RNA-guided nucleases that include inserts encoded in part by cellular genomic or other sequences recognized by guide RNAs.
Claims31
-30. (canceled)
A nucleic acid molecule comprising a nucleic acid sequence encoding a Cpf1 protein, wherein the nucleic acid sequence comprises a eukaryotic nucleic acid sequence that
- a) is at least 17 nucleotides in length;
- and
- b) comprises or is adjacent to a protospacer adjacent motif (PAM) that is recognized by the Cpf1 protein.
The nucleic acid molecule of claim 1, wherein the Cpf1 protein comprises an amino acid insertion, relative to SEQ ID NO:
- 13, at a position selected from the group consisting of
- a) between amino acid positions 147 and 148;
- b) anywhere between amino acid positions 484 and 492,
- c) anywhere between amino acid positions 568 and 590,
- d) anywhere between amino acid positions 795 and 855,
- e) anywhere between amino acid positions 1131 and 1140, and
- f) anywhere between amino acid positions 1160 and 1173.
A method of altering a target site in a cell comprising delivering to the cell a transiently active genome editing system, the transiently expressed genome editing system comprising:
- a) a guide RNA (gRNA) comprising a targeting domain that is complementary to a eukaryotic nucleic acid sequence;
- and
- b) an engineered Cpf1 protein encoded by a nucleic acid comprising the eukaryotic nucleotide sequence and a protospacer adjacent motif (PAM), wherein the PAM is recognized by the Cpf1 protein and is within or adjacent to the eukaryotic nucleotide sequence.
The method of claim 19, wherein the Cpf1/gRNA complex is adapted to cleave the nucleic acid encoding the engineered Cpf1 protein.
The method of claim 19, wherein the Cpf1/gRNA complex is adapted to cleave both the nucleic acid encoding the engineered Cpf1 protein and the target site in the cell.
The method of claim 18, wherein the engineered Cpf1 protein comprises an amino acid insertion or substitution that is at least partially encoded by the eukaryotic nucleotide sequence.
The method of claim 18, wherein the engineered Cpf1 protein has at least about 80% nuclease activity of a wild-type Cpf1 protein.
The method of claim 18, wherein the engineered Cpf1 protein comprises a polypeptide comprising the sequence of G-(X)6-10-G.
The method of claim 18, wherein the engineered Cpf1 protein comprises an amino acid insertion, relative to SEQ ID NO:
- 13, at a position selected from the group consisting of
- a) between amino acid positions 147 and 148,
- b) anywhere between amino acid positions 484 and 492,
- c) anywhere between amino acid positions 568 and 590,
- d) anywhere between amino acid positions 795 and 855,
- e) anywhere between amino acid positions 1131 and 1140, and
- f) anywhere between amino acid positions 1160 and 1173.
The method of claim 18, wherein the engineered Cpf1 protein comprises an amino acid insertion relative to SEQ ID NO:
- 13 at or near the N-terminus of the Cpf1 protein.
The method of claim 18, wherein the engineered Cpf1 protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:
- 13.
The method of claim 18, wherein the engineered Cpf1 protein comprises an amino acid insertion, relative to SEQ ID NO:
- 14, at a position selected from the group consisting of:
- a) between nucleic acid positions 441 and 442,
- b) anywhere between nucleic acid positions 1452 and 1474,
- c) anywhere between nucleic acid positions 1704 and 1768,
- d) anywhere between nucleic acid positions 2385 and 2563,
- e) anywhere between nucleic acid positions 3393 and 3418, and
- f) anywhere between nucleic acid positions 3480 and 3517;
- wherein the insertion does not alter the reading frame of the isolated nucleic acid.
Dependencies are inferred from the claim text.