Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates
Year: 2025
Type: article
Abstract: 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.
Source: Nature Communications
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $6,990
Funders: National Science Foundation, Howard Hughes Medical Institute, U.S. Department of Health and Human Services, Achievement Rewards for College Scientists Foundation, National Institutes of Health, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases
OpenAlex ID: W4408877609