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US 11,236,313 B2Granted
Cas9 fusion molecules, gene editing systems, and methods of use thereof
AssigneeEditas Medicine, Inc.
InventorsCecilia Cotta-Ramusino, Hariharan Jayaram, John Anthony Zuris
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Abstract
Disclosed herein are enzymatically active Cas9 (eaCas9) fusion molecules, comprising an eaCas9 molecule linked, e.g., covalently or non-covalently, to a template nucleic acid; gene editing systems comprising the eaCas9 fusion molecules, and methods of use thereof.
Claims14
An enzymatically active Cas9 (eaCas9) fusion molecule comprising an eaCas9 molecule covalently linked to a template nucleic acid by a polypeptide linker.
A gene editing system, comprising
- at least one enzymatically active Cas9 (eaCas9) fusion molecule comprising an eaCas9 molecule covalently linked to a template nucleic acid by a polypeptide linker, and
- at least one gRNA molecule.
A gene editing system, comprising
- (i) a first eaCas9 fusion molecule, wherein the first eaCas9 fusion molecule comprises a first Cas9 nickase molecule covalently linked to a template nucleic acid by a polypeptide linker;
- a first gRNA molecule;
- a second eaCas9 fusion molecule, wherein the second eaCas9 fusion molecule comprises a second Cas9 nickase molecule covalently linked to the template nucleic acid by a polypeptide linker;
- and
- a second gRNA molecule;
- or
- (ii) a first eaCas9 fusion molecule, wherein the first eaCas9 fusion molecule comprises a first Cas9 nickase molecule covalently linked to a first template nucleic acid by a polypeptide linker;
- a first gRNA molecule;
- a second eaCas9 fusion molecule, wherein the second eaCas9 fusion molecule comprises a second Cas9 nickase molecule covalently linked to a second template nucleic acid by a polypeptide linker;
- and
- a second gRNA molecule.
8 more elements
A method of modifying a target nucleic acid in a cell, the method comprising:
- contacting the cell with a first gRNA molecule;
- a first eaCas9 molecule;
- a second gRNA molecule;
- and a second eaCas9 molecule;
- wherein at least one of the first and second eaCas9 molecule is covalently linked to a template nucleic acid by a polypeptide linker,
- wherein the first gRNA molecule and the first eaCas9 molecule associate with the target nucleic acid and generate a first single strand cleavage event on a first strand of the target nucleic acid;
- wherein the second gRNA molecule and the second eaCas9 molecule associate with the target nucleic acid and generate a second single strand cleavage event on a second strand of the target nucleic acid, thereby forming a double strand break having a first overhang and a second overhang;
- and wherein the first overhang and the second overhang in the target nucleic acid are repaired by gene correction using the template nucleic acid.
5 more elements
Dependencies are inferred from the claim text.