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Article

Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis.

Nature structural & molecular biology - 1 Apr 2018

Singh Digvijay, Wang Yanbo, Mallon John, Yang Olivia, Fei Jingyi, Poddar Anustup, Ceylan Damon, Bailey Scott, Ha Taekjip

Abstract excerpt

Cas9 (from Streptococcus pyogenes) in complex with a guide RNA targets complementary DNA for cleavage. Here, we developed a single-molecule FRET analysis to study the mechanisms of specificity enhancement of two engineered Cas9s (eCas9 and Cas9-HF1). A DNA-unwinding assay showed that mismatches affect cleavage reactions through rebalancing the unwinding-rewinding equilibrium. Increasing PAM-distal mismatches...

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Identifiers

PMID: 29622787
DOI: 10.1038/s41594-018-0051-7
PMCID: PMC6195204

Genes

PAM

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