Kiernan Kaitlyn A, Taylor David W
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
Institute of Science and Technology Austria, Am Campus 1, A-3400, Klosterneuburg, Austria.
Nat Commun. 2025 Jul 1;16(1):5681. doi: 10.1038/s41467-025-60668-7.
While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Cas9 can eventually be removed from the product by extrinsic factors, such as translocating polymerases, but the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and promote faster enzyme turnover. Using kinetics-guided cryo-EM, we examine the conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.
虽然使用最广泛的CRISPR-Cas酶是化脓性链球菌的Cas9核酸内切酶（Cas9），但它表现出单轮酶动力学，这导致其在产物DNA上的停留时间较长。这阻碍了DNA修复机制的作用，并且在CRISPR-Cas9基因编辑过程中成为一个主要瓶颈。Cas9最终可以通过外部因素（如易位聚合酶）从产物中去除，但切割后导致Cas9解离的机制仍知之甚少。在这里，我们采用截短的引导RNA作为一种策略，以削弱PAM远端核酸相互作用并促进更快的酶周转。利用动力学引导的冷冻电镜，我们研究了多轮Cas9的构象变化，包括Cas9从产物DNA上解离的首张详细快照。我们发现，虽然PAM远端产物在切割后从Cas9上解离，但PAM近端产物的紧密结合直接抑制了新靶点的重新结合。我们的工作为Cas9为何作为单轮酶提供了直接证据，并将指导未来的Cas9工程研究。
