Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis

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.

Automated summary

This study utilized single-molecule FRET assay to investigate the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants, revealing a strong correlation between their conformational structure and nuclease activity. Quantitative analysis showed that these engineered Cas9 variants exhibit a more rapid decrease in cleavage-competent state with increasing base-pair mismatch, leading to enhanced target specificity. The critical specificity enhancement step was found to be activated during DNA:RNA heteroduplexation for some variants, highlighting the importance of conformational dynamics in Cas9 target specificity.