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

Abstract

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 facilitates rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily with mismatches. The intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. Engineered Cas9s require approximately one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity by inhibiting stable DNA binding to partially matching sequences, making DNA unwinding more sensitive to mismatches and slowing down the intrinsic cleavage reaction.

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

“…A variety of experimental systems are widely being used to investigate the off‐target effects of sgRNAs and they show different outcomes regarding the extent of off‐targets . Many algorithms have been applied for the prediction of off‐target effects and many Cas9 proteins with high specificity have successfully been produced for minimizing Cas9 promiscuity . However, sequencing‐based approaches have been widely used to experimentally validate a variety of Cas9 off‐targets that are not fully explained by such algorithms, raising an interesting question about mechanisms of binding of Cas9 complexes and cleavage of off‐targets .…”

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

“…Several Cas9 variants have been engineered (Engineered Cas9; EngCas9) to improve cleavage specificity [10][11][12][13][14][15][16][17] . Stable binding by Cas9s requires as few as 9-10 bp between the PAM-proximal end of the protospacer and the gRNA, but cle avage re quire s more bps (~16 for WT Cas9 and ~17-18 for EngCas9s) 5,7,18 . We previously showed, using a single molecule FRET unwinding assay, (Fig.…”

“…1a) that the DNA in Cas9-RNA-DNA undergoes internal unwinding-rewinding dynamics and the unwound state is a critical checkpoint required for cleavage. The intrinsic rate of cleavage (kc, int) has been used to refer to the rate of cleavage from the unwound state 15,18 . The PAM-distal mismatches reduce the lifetime and extent of the unwound state, thereby delaying or preventing cleavage 18 .…”

“…The intrinsic rate of cleavage (kc, int) has been used to refer to the rate of cleavage from the unwound state 15,18 . The PAM-distal mismatches reduce the lifetime and extent of the unwound state, thereby delaying or preventing cleavage 18 . EngCas9s; eCas9 10 , and Cas9-HF1 19 , have higher cleavage specificity in part because their mutations make it difficult to unwind DNA when the sequence match is not perfect 18 .…”

“…The PAM-distal mismatches reduce the lifetime and extent of the unwound state, thereby delaying or preventing cleavage 18 . EngCas9s; eCas9 10 , and Cas9-HF1 19 , have higher cleavage specificity in part because their mutations make it difficult to unwind DNA when the sequence match is not perfect 18 . These variants also lower kc, int thereby making it less likely that cleavage can occur from transiently unwound states 18 .…”