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  • Stepwise DNA unwinding gates TnpB genome-editing activity. bioRxiv : the preprint server for biology Zhou, Z., Saffarian-Deemyad, I., Shi, H., Weiss, T., Ur-Rehman, M. M., Vohra, K., Skopintsev, P., Yoon, P. H., Trinidad, M. I., Langeberg, C. J., Kamalu, M., Amerasekera, J., Doherty, E. E., Aris, K. D., Al-Sayyad, N., Thornton, B. W., Weissman, R. F., Wasko, K. M., Esain-Garcia, I., DeTurk, E. C., Savage, D. F., Jacobsen, S. E., Bryant, Z., Doudna, J. A. 2026

    Abstract

    TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through formation of a partially unwound intermediate state to a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, Ymu1-WFR, stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in vivo. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.

    View details for DOI 10.64898/2026.01.09.698545

    View details for PubMedID 41542533

    View details for PubMedCentralID PMC12803034

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

    Abstract

    RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical, and cell-based assays on Streptococcus pyogenes Cas9 (SpyCas9) variants with wide-ranging genome-editing efficiencies that differ in PAM-binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome-editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome-editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.

    View details for DOI 10.1016/j.molcel.2025.03.024

    View details for PubMedID 40273916

  • 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

  • Ancestral RNA-guided TnpB nuclease interrogates DNA in discrete supercoiling-sensitive steps Saffarian-Deemyad, I., Zhou, Z., Aris, K., Shi, H., Al-Sayyad, N., Doudna, J. A., Bryant, Z. CELL PRESS. 2025

    View details for Web of Science ID 001510145400196

  • 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