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

Author

Listed:
  • Kevin D. P. Aris

    (Stanford University
    Stanford University)

  • Joshua C. Cofsky

    (University of California, Berkeley
    Harvard Medical School)

  • Honglue Shi

    (University of California, Berkeley
    University of California, Berkeley)

  • Noor Al-Sayyad

    (Stanford University
    Stanford University)

  • Ivan E. Ivanov

    (Stanford University
    Stanford University
    Chan Zuckerberg Biohub)

  • Ashwin Balaji

    (Stanford University)

  • Jennifer A. Doudna

    (University of California, Berkeley
    University of California, Berkeley
    University of California, Berkeley)

  • Zev Bryant

    (Stanford University
    Stanford University Medical Center)

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.

Suggested Citation

  • Kevin D. P. Aris & Joshua C. Cofsky & Honglue Shi & Noor Al-Sayyad & Ivan E. Ivanov & Ashwin Balaji & Jennifer A. Doudna & Zev Bryant, 2025. "Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-57703-y
    DOI: 10.1038/s41467-025-57703-y
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