Noor Al-Sayyad

All Publications

• 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

• 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