A rational engineering strategy for structural dynamics modulation enables target specificity enhancement of the Cas9 nuclease.

Structural dynamics of an enzyme plays a crucial role in enzymatic activity and substrate specificity, yet rational engineering of the dynamics for improved enzymatic properties remains a challenge. Here, we present a new biochemical strategy of intermediate state stabilization that modulates the multistep dynamic mechanisms of enzyme reactions to improve substrate specificity. We employ this strategy to enhance CRISPR-Cas9 nuclease specificity. By incorporating positively charged residues into the noncatalytic REC2 domain of Cas9, we stabilize the REC2-DNA interaction that forms exclusively in a catalytically inactive intermediate conformation of the Cas9 complex. This enables off-target trapping in the inactive conformation and thus reduces off-target cleavage in human cells. Furthermore, we combine the REC2 modification with mutations in previous rational variants, leading to the development of a combinational variant named Correct-Cas9, which connotes "combined with rationally engineered REC-Two" Cas9. Assessed by high-throughput analysis at thousands of target sequences, Correct-Cas9 exhibits increased target specificity compared to its parental variants, demonstrating a synergy between our strategy and previous rational approaches. Our method of intermediate state stabilization, either alone or combined with conventional approaches, could be applied to various nucleic acid-processing enzymes that undergo conformational changes upon target binding, to enhance their target specificity effectively.