Sung Keewon, Jung Youngri, Kim Nahye, Kim Yong-Woo, Kim Hyongbum Henry, Kim Seong Keun, Bae Sangsu
Department of Chemistry, Seoul National University, Seoul 08826, South Korea.
Research Institute of Basic Sciences, Seoul National University, Seoul 08826, South Korea.
Nucleic Acids Res. 2025 Jun 20;53(12). doi: 10.1093/nar/gkaf535.
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.
酶的结构动力学在酶活性和底物特异性中起着关键作用，然而，通过合理设计动力学来改善酶的性质仍然是一个挑战。在此，我们提出一种新的中间态稳定化生化策略，该策略可调节酶反应的多步动力学机制以提高底物特异性。我们运用这一策略来增强CRISPR-Cas9核酸酶的特异性。通过将带正电荷的残基引入Cas9的非催化REC2结构域，我们稳定了REC2与DNA的相互作用，这种相互作用仅在Cas9复合物的催化无活性中间构象中形成。这使得脱靶在无活性构象中被捕获，从而减少了在人类细胞中的脱靶切割。此外，我们将REC2修饰与先前合理设计变体中的突变相结合，从而开发出一种名为Correct-Cas9的组合变体，其含义为“与合理设计的REC2相结合的”Cas9。通过对数千个靶序列进行高通量分析评估，Correct-Cas9与其亲本变体相比，显示出更高的靶标特异性，这表明我们的策略与先前的合理方法之间具有协同作用。我们的中间态稳定化方法，无论是单独使用还是与传统方法相结合，都可应用于各种在结合靶标时会发生构象变化的核酸加工酶，以有效提高它们的靶标特异性。
