ATPase facilitates PlmCas12e-mediated gene editing through conformational modulation
Abstract
CRISPR-Cas systems represent powerful and versatile tools for genome engineering, yet the editing efficacy of compact effectors such as Cas12e is often constrained by the intrinsic energy barriers associated with conformational activation. Here, we develop a systematic screening strategy to evaluate the performance of various ATPases fused to SpCas9 and PlmCas12e effectors using diverse linker architectures, aiming to overcome conformational activation barriers through ATPase-driven remodeling. One lead architecture, PlmCas12e fused with DnaA at the N-terminus via a rigid linker, demonstrates significantly enhanced editing efficiencies across exogenous and endogenous targets in HEK293T cells. In vitro biochemical assays further suggest enhanced intrinsic catalytic activity of the fusion variant. Molecular dynamics simulations show that DnaA-fused variant exhibits a more confined Free Energy Landscape (FEL) and reduced Root Mean Square Fluctuation (RMSF) in critical domains. These findings support a model in which DnaA facilitates conformational pre-organization, thereby lowering the entropic cost of activation for PlmCas12e to transition into a catalytically active state. Overall, our study not only provides a robustly enhanced PlmCas12e variant for genome editing but also highlights conformational modulation as a strategy for engineering CRISPR effectors.
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