Engineered nucleases and methods of use thereof
AssigneeMassachusetts Institute of Technology
InventorsVIKASH PAL SINGH CHAUHAN, PHILLIP A. SHARP, ROBERT LANGER
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Abstract
The present disclosure provides a method of editing a genome in a cell including exposing the cell to an engineered Cas nuclease comprising one or more mutations within the DNA binding cleft of the Cas nuclease, wherein exposure to the engineered Cas nuclease decreases, inhibits, or prevents non-homologous end joining (NHEJ) in the cell, and wherein exposure to the engineered Cas nuclease increases one or more homology-driven repair pathways within the cell. The mutant Cas nuclease is also disclosed herein.
Claims29
A method of editing a genome in a cell, the method comprising:
- exposing the cell to an engineered Cas nuclease comprising one or more mutations within the DNA binding cleft of the Cas nuclease,
- wherein exposure to the engineered Cas nuclease decreases, inhibits, or prevents an indel-producing DNA repair pathway in the cell, and
- wherein exposure to the engineered Cas nuclease increases one or more precise editing repair pathways within the cell.
A method of precisely editing the genome of a non-dividing cell, the method comprising administering to the cell an agent capable of inhibiting or preventing non-homologous end joining (NHEJ) and increasing homology-driven repair (HDR).
An engineered Cas nuclease variant comprising two or more amino acid substitutions, mutations, or deletions in the DNA binding cleft such that the engineered Cas nuclease variant predominantly engages a homology-driven DNA repair pathway.
A method of editing a genome in a cell, the method comprising:
- exposing the cell to an engineered Cas nuclease comprising one or more mutations within the DNA binding cleft of the Cas nuclease,
- wherein the engineered Cas nuclease is fused or otherwise associated with a polymerase to form a prime editor,
- wherein exposure to the engineered Cas nuclease increases precise genome editing initiated by action of the associated polymerase, and
- wherein exposure to the engineered Cas nuclease decreases, inhibits, or prevents byproduct indel formation in the cell.
An engineered Cas nuclease variant comprising one or more amino acid substitutions, mutations, or deletions in the DNA binding cleft, wherein the engineered Cas nuclease is fused or otherwise associated with a polymerase to form a prime editor.
Dependencies are inferred from the claim text.