Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates

Kevin D. P. Aris, Joshua C. Cofsky, Honglue Shi, Noor Al-Sayyad, Ivan E. Ivanov, Ashwin Balaji, Jennifer A. Doudna and Zev Bryant ()
Additional contact information
Kevin D. P. Aris: Stanford University
Joshua C. Cofsky: University of California, Berkeley
Honglue Shi: University of California, Berkeley
Noor Al-Sayyad: Stanford University
Ivan E. Ivanov: Stanford University
Ashwin Balaji: Stanford University
Jennifer A. Doudna: University of California, Berkeley
Zev Bryant: Stanford University

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: 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.

Date: 2025
References: Add references at CitEc
Citations:

Downloads: (external link)
https://www.nature.com/articles/s41467-025-57703-y Abstract (text/html)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-57703-y

Ordering information: This journal article can be ordered from
https://www.nature.com/ncomms/

DOI: 10.1038/s41467-025-57703-y

Access Statistics for this article

Nature Communications is currently edited by Nathalie Le Bot, Enda Bergin and Fiona Gillespie

More articles in Nature Communications from Nature
Bibliographic data for series maintained by Sonal Shukla () and Springer Nature Abstracting and Indexing ().