Department of Bioengineering, University of California Riverside, 900 University Avenue, Riverside, CA, 52512, USA.
Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland.
Nat Commun. 2024 Feb 17;15(1):1473. doi: 10.1038/s41467-024-45762-6.
CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:target strand duplex toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the target strand within the RuvC core by bending inward. Understanding of this critical process underlying Cas12a activity will enrich fundamental knowledge and facilitate further engineering strategies for genome editing.
CRISPR-Cas12a 是一种强大的 RNA 导向基因组编辑系统，它通过连续的机制利用其单一的 RuvC 核酸酶结构域产生双链 DNA 断裂，其中初始非靶链的切割后接着是靶链的切割。目前尚不清楚空间上遥远的 DNA 靶链如何朝向 RuvC 催化核心迁移。在这里，连续数十微秒长的分子动力学和自由能模拟揭示了位于 RuvC 结构域内的 α-螺旋盖在 DNA 靶链迁移过程中起着关键作用，它通过锚定 crRNA:靶链双链并引导靶链朝向 RuvC 核心，这也得到了 DNA 切割实验的证实。在这种机制中，REC2 结构域将 crRNA:靶链双链推向酶的核心，而 Nuc 结构域通过向内弯曲来帮助靶链在 RuvC 核心内弯曲和适应。对 Cas12a 活性的这一关键过程的理解将丰富基础知识，并为基因组编辑的进一步工程策略提供便利。
