Methods and compositions for blocking off-target nucleic acids from cleavage by CRISPR proteins
AssigneeAgilent Technologies, Inc.
InventorsChong Wing Yung, Andrew Kennedy
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Abstract
This invention relates to reagents and methods for increasing specificity and efficiency of genome editing by CRISPR associated (Cas) protein systems, more particularly by Cas:guide RNA complexes, by blocking off-target nucleic acids from cleavage by Cas:guide RNA complexes.
Claims20
A composition or kit for gene editing comprising:
- at least one Cas protein, or at least one nucleic acid encoding a Cas protein;
- a first guide RNA, wherein the first guide RNA and at least one of said Cas protein are adapted to form a first Cas:guide RNA complex, wherein the first guide RNA comprises a targeting guide sequence substantially complementary to a target nucleic acid sequence;
- a second guide RNA, wherein the second guide RNA and at least one of said Cas protein are adapted to form a second Cas:guide RNA complex, wherein the second guide RNA comprises a blocking guide sequence substantially complementary (i) to an off-target nucleic acid sequence;
- (ii) to a nucleic acid sequence comprising a portion of an off-target nucleic acid sequence and a flanking nucleic acid sequence adjacent to the portion;
- or (iii) to a nucleic acid sequence at a location relative to an off-target nucleic acid sequence such that the second Cas:guide RNA complex blocks the first Cas:guide RNA complex from the off-target nucleic acid sequence;
- wherein the second Cas:guide RNA complex is substantially inactive at cleaving the off-target nucleic acid sequence and blocks the off-target nucleic acid sequence from the first Cas:guide RNA complex.
A collection or library comprising two or more guide RNAs, wherein a first guide RNA comprises a first guide sequence substantially complementary to a target nucleic acid sequence, and a second guide RNA comprises a blocking guide sequence substantially complementary (i) to an off-target nucleic acid sequence;
- (ii) to a portion of an off-target nucleic acid sequence and a nucleic acid adjacent to the portion;
- or (iii) to a nucleic acid sequence at a location relative to an off-target nucleic acid sequence such that a complex of the second guide RNA with a Cas protein blocks the off-target nucleic acid sequence.
A vector encoding a first guide RNA comprising a first guide sequence substantially complementary to a target nucleic acid sequence, and a second guide RNA comprising a blocking guide sequence substantially complementary (i) to an off-target nucleic acid sequence;
- (ii) to a portion of an off-target nucleic acid sequence and a nucleic acid adjacent to the portion;
- or (iii) to a nucleic acid sequence at a location relative to an off-target nucleic acid sequence such that a complex of the second guide RNA with a Cas protein blocks the off-target nucleic acid sequence.
Dependencies are inferred from the claim text.