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; Zev Bryant

doi:10.1038/s41467-025-57703-y

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

Nat Commun. 2025 Mar 26;16(1):2939. doi: 10.1038/s41467-025-57703-y.

Authors

Kevin D P Aris^{1

2}, Joshua C Cofsky^{3

4}, Honglue Shi^{5

6}, Noor Al-Sayyad^{2

7}, Ivan E Ivanov^{2

8

9}, Ashwin Balaji¹, Jennifer A Doudna^{3

5

6}, Zev Bryant^{10

11}

Affiliations

¹ Biophysics Program, Stanford University, Stanford, CA, USA.
² Department of Bioengineering, Stanford University, Stanford, CA, USA.
³ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
⁴ Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
⁵ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA.
⁶ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
⁷ Department of Physics, Stanford University, Stanford, CA, USA.
⁸ Department of Chemical Engineering, Stanford University, Stanford, CA, USA.
⁹ Chan Zuckerberg Biohub, San Francisco, CA, USA.
¹⁰ Department of Bioengineering, Stanford University, Stanford, CA, USA. zevry@stanford.edu.
¹¹ Department of Structural Biology, Stanford University Medical Center, Stanford, CA, USA. zevry@stanford.edu.

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.

MeSH terms

Bacterial Proteins* / chemistry
Bacterial Proteins* / genetics
Bacterial Proteins* / metabolism
CRISPR-Associated Proteins* / chemistry
CRISPR-Associated Proteins* / genetics
CRISPR-Associated Proteins* / metabolism
CRISPR-Cas Systems / genetics
DNA* / chemistry
DNA* / genetics
DNA* / metabolism
DNA, Superhelical* / chemistry
DNA, Superhelical* / genetics
DNA, Superhelical* / metabolism
Endodeoxyribonucleases* / chemistry
Endodeoxyribonucleases* / genetics
Endodeoxyribonucleases* / metabolism
Kinetics
R-Loop Structures*
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

CRISPR-Associated Proteins
Cas12a protein
DNA, Superhelical
RNA, Guide, CRISPR-Cas Systems
DNA
Endodeoxyribonucleases
Bacterial Proteins

Abstract

MeSH terms

Substances

Grants and funding