한빛사 논문
So Young Bak1,†, Youngri Jung2,†, Jinho Park1,†, Keewon Sung1,†, Hyeon-Ki Jang2, Sangsu Bae2,* and Seong Keun Kim1,*
1Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea and 2Department of Chemistry and Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Republic of Korea
*To whom correspondence should be addressed.
†The authors wish it to be known that, in their opinion, the first four authors should be regarded as Joint First Authors.
Abstract
There have been many engineered Cas9 variants that were developed to minimize unintended cleavage of off-target DNAs, but detailed mechanism for the way they regulate the target specificity through DNA:RNA heteroduplexation remains poorly understood. We used single-molecule FRET assay to follow the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants with respect to on-target and off-target DNAs. Just like wild-type Cas9, these engineered Cas9 variants exhibit a strong correlation between their conformational structure and nuclease activity. Compared with wild-type Cas9, the fraction of the cleavage-competent state dropped more rapidly with increasing base-pair mismatch, which gives rise to their enhanced target specificity. We proposed a reaction model to quantitatively analyze the degree of off-target discrimination during the successive process of R-loop expansion. We found that the critical specificity enhancement step is activated during DNA:RNA heteroduplexation for evoCas9 and HypaCas9, while it occurs in the post-heteroduplexation stage for Cas9-HF1, eCas9, and Sniper-Cas9. This study sheds new light on the conformational dynamics behind the target specificity of Cas9, which will help strengthen its rational designing principles in the future.
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