Aris Kevin D P, Cofsky Joshua C, Shi Honglue, Al-Sayyad Noor, Ivanov Ivan E, Balaji Ashwin, Doudna Jennifer A, Bryant Zev
Biophysics Program, Stanford University, Stanford, CA, USA.
Department of Bioengineering, Stanford University, Stanford, CA, USA.
Nat Commun. 2025 Mar 26;16(1):2939. doi: 10.1038/s41467-025-57703-y.
The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5 bp initial RNA-DNA hybridization and a ~17 bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity.
DNA结合与切割的序列特异性和可编程性使得CRISPR-Cas12a在基因工程中得到了广泛应用。作为一种RNA引导的CRISPR核酸内切酶，Cas12a通过形成三链R环结构与一段20个碱基对（bp）的DNA片段结合，其中引导RNA与DNA靶标杂交。在此，我们使用单分子扭矩光谱技术，以碱基对分辨率研究两种广泛使用的Cas12a直系同源物R环形成的动力学和力学过程。我们直接观察到对应于约5 bp初始RNA-DNA杂交以及R环完成前约17 bp中间体的动力学中间体，随后是延伸超过20 bp R环的瞬时DNA解旋。R环形成的复杂多状态格局取决于直系同源物，并受靶标序列、错配和DNA超螺旋的影响。一个四态动力学模型捕捉了Cas12a R环动力学的基本特征，并为理解Cas12a的活性和特异性提供了一个生物物理框架。
