PT  - JOURNAL ARTICLE
AU  - Digvijay Singh
AU  - Yanbo Wang
AU  - John Mallon
AU  - Olivia Yang
AU  - Jingyi Fei
AU  - Anustup Poddar
AU  - Damon Ceylan
AU  - Scott Bailey
AU  - Taekjip Ha
TI  - Mechanisms of improved specificity of engineered Cas9s revealed by single molecule analysis
AID  - 10.1101/192724
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 192724
4099  - http://biorxiv.org/content/early/2017/09/22/192724.short
4100  - http://biorxiv.org/content/early/2017/09/22/192724.full
AB  - In microbes, CRISPR-Cas systems provide adaptive immunity against invading genetic elements. Cas9 in complex with a guide-RNA targets complementary DNA for cleavage and has been repurposed for wide-ranging biological applications. New Cas9s have been engineered (eCas9 and Cas9-HF1) to improve specificity, but how they help reduce off-target cleavage is not known. Here, we developed single molecule DNA unwinding assay to show that sequence mismatches affect cleavage reactions through rebalancing the internal unwinding/rewinding equilibrium. Increasing PAM-distal mismatches facilitate rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily upon mismatch detection. Intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. DNA interrogation experiments showed that engineered Cas9s require about one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity (1) by inhibiting stable DNA binding to partially matching sequences, (2) by making DNA unwinding more sensitive to mismatches, and (3) by slowing down intrinsic cleavage reaction.