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

Journal Article · Wed Mar 26 00:00:00 UTC 2025 · Nature Communications

DOI:https://doi.org/10.1038/s41467-025-57703-y· OSTI ID:2551794

Aris, Kevin D. P. ^[1]; Cofsky, Joshua C. ^[2]; Shi, Honglue ^[3]; Al-Sayyad, Noor ^[1]; Ivanov, Ivan E. ^[4]; Balaji, Ashwin ^[1]; Doudna, Jennifer A. ^[3]; Bryant, Zev ^[4]

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1. Stanford University, CA (United States)
2. University of California, Berkeley, CA (United States); Harvard Medical School, Boston, MA (United States)
3. University of California, Berkeley, CA (United States)
4. Stanford University, CA (United States); Chan Zuckerberg Biohub, San Francisco, CA (United States)

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.

• Research Organization: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
• Sponsoring Organization: National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC), Biological and Environmental Research (BER)
• Grant/Contract Number: AC02-05CH11231
• OSTI ID: 2551794
• Journal Information: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 16; ISSN 2041-1723
• Publisher: Nature Publishing Group
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES