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All Publications

  • Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates. Nature communications Aris, K. D., Cofsky, J. C., Shi, H., Al-Sayyad, N., Ivanov, I. E., Balaji, A., Doudna, J. A., Bryant, Z. 2025; 16 (1): 2939

    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 ~5bp initial RNA-DNA hybridization and a ~17bp 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.

    View details for DOI 10.1038/s41467-025-57703-y

    View details for PubMedID 40133266

  • Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing Shi, H., Al-Sayyad, N., Wasko, K., Trinidad, M. I., Doherty, E. E., Vohra, K., Boger, R., Colognori, D., Cofsky, J., Skopintsev, P., Bryant, Z., Doudna, J. A. CELL PRESS. 2025: 90A

    View details for Web of Science ID 001440845200016

  • Torque spectroscopy of molecular friction: From basepair stacking to Cas9 Al-Sayyad, N., Starr, C. H., Ivanov, I. E., Cofsky, J., Aris, K., Shi, H., Doudna, J. A., Bryant, Z. CELL PRESS. 2024: 500A

    View details for Web of Science ID 001194120702826

  • Characterizing dissociative motion in time-resolved x-ray scattering from gas-phase diatomic molecules PHYSICAL REVIEW A Ware, M. R., Glownia, J. M., Al-Sayyad, N., O'Neal, J. T., Bucksbaum, P. H. 2019; 100 (3)

    View details for DOI 10.1103/PhysRevA.100.033413

    View details for Web of Science ID 000486625200003

  • Observing Molecular Dynamics Using Frequency Resolved X-ray Scattering Glownia, J. M., Ware, M., Al-Sayyad, N., Bucksbaum, P., IEEE IEEE. 2019

    View details for Web of Science ID 000630002701308