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

ABSTRACT: 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

SUBMITTER: Sung K 

PROVIDER: S-EPMC12204706 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature