Back to results
US 2019/0161743 A1Published application
Self-Targeting Guide RNAs in CRISPR System
AssigneePresident and Fellows of Harvard College
InventorsGeorge M. Church, Reza Kalhor, Prashant G. Mali
Life of this patent
Estimating the term…
Abstract
CRISPR/Cas9 methods are provided where a guide RNA is engineered to self-target and inactivate a nucleic acid encoding the guide RNA itself.
Claims67
A method of targeting a nucleic acid encoding a guide RNA in a cell comprising
- introducing into the cell a first foreign nucleic acid encoding a guide RNA sequence including a spacer sequence and a protospacer adjacent motif (PAM) adjacent to the spacer sequence, wherein the spacer sequence is complementary to a protospacer sequence in the first foreign nucleic acid and to a protospacer sequence in a target nucleic acid sequence of the genomic DNA,
- introducing into the cell a second foreign nucleic acid encoding a Cas9 protein,
- wherein the guide RNA sequence and the Cas9 protein are expressed, and
- wherein the guide RNA sequence and the Cas9 protein co-localize to the first foreign nucleic acid and the Cas9 protein binds or cleaves the first foreign nucleic acid sequence in a site specific manner.
1 more element
A method of targeting a nucleic acid encoding a guide RNA in vitro comprising
- providing a first foreign nucleic acid encoding a guide RNA sequence including a spacer sequence and a protospacer adjacent motif (PAM) adjacent to the spacer sequence, wherein the spacer sequence is complementary to a protospacer sequence in the first foreign nucleic acid,
- providing a second foreign nucleic acid encoding a Cas9 protein,
- wherein the guide RNA sequence and the Cas9 protein are expressed, and
- wherein the guide RNA sequence and the Cas9 protein co-localize to the first foreign nucleic acid and the Cas9 protein binds or cleaves the first foreign nucleic acid sequence in a site specific manner.
1 more element
A cell comprising
- a first foreign nucleic acid encoding a guide RNA sequence including a spacer sequence and a protospacer adjacent motif (PAM) adjacent to the spacer sequence, wherein the spacer sequence is complementary to a protospacer sequence in the first foreign nucleic acid and a protospacer sequence in a target nucleic acid sequence of the genomic DNA,
- a second foreign nucleic acid encoding a Cas9 protein,
- wherein the guide RNA sequence and the Cas9 protein are expressed, and
- wherein the guide RNA sequence and the Cas9 protein co-localize to the first foreign nucleic acid and the Cas9 protein binds or cleaves the first foreign nucleic acid sequence in a site specific manner.
1 more element
An in vitro CRISPR system comprising
- a first foreign nucleic acid encoding a guide RNA sequence including a spacer sequence and a protospacer adjacent motif (PAM) adjacent to the spacer sequence, wherein the spacer sequence is complementary to a protospacer sequence in the first foreign nucleic acid,
- a second foreign nucleic acid encoding a Cas9 protein,
- wherein the guide RNA sequence and the Cas9 protein are expressed, and
- wherein the guide RNA sequence and the Cas9 protein co-localize to the first foreign nucleic acid and the Cas9 protein binds or cleaves the first foreign nucleic acid sequence in a site specific manner.
1 more element
A method of targeting a nucleic acid sequence using a CRISPR system comprising
- providing a first foreign nucleic acid encoding a guide RNA sequence including a spacer sequence complementary to a protospacer sequence in the nucleic acid sequence,
- providing a second foreign nucleic acid encoding a Cas9 protein,
- wherein the guide RNA sequence and the Cas9 protein are expressed,
- wherein the guide RNA sequence and the Cas9 protein co-localize to the nucleic acid sequence and the Cas9 protein binds or cleaves the nucleic acid sequence in a site specific manner, and
- wherein the rate at which the guide RNA regulates the binding or cleavage of the nucleic acid sequence can be controlled.
2 more elements
Dependencies are inferred from the claim text.