Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9

Hibshman GN, Bravo JPK, Hooper MM, Dangerfield TL, Zhang H, Finkelstein IJ, Johnson KA, Taylor DW. 2024. Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9. Nature Communications. 15, 3663.

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Journal Article | Published | English

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Author
Hibshman, Grace N.; Bravo, JackISTA ; Hooper, Matthew M.; Dangerfield, Tyler L.; Zhang, Hongshan; Finkelstein, Ilya J.; Johnson, Kenneth A.; Taylor, David W.
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Abstract
CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.
Publishing Year
Date Published
2024-04-30
Journal Title
Nature Communications
Acknowledgement
We thank I. Stohkendl in the Taylor group for insightful discussions. This work was supported in part by Welch Foundation grants F-1808 (to I.J.F.), and F-1938 (to D.W.T.), the National Institutes of Health R01GM124141 (to I.J.F.), R01AI110577 (to K.A.J.), and R35GM138348 (to D.W.T.), and a Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation Medical Research Grant (to D.W.T.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Volume
15
Article Number
3663
eISSN
IST-REx-ID

Cite this

Hibshman GN, Bravo JPK, Hooper MM, et al. Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9. Nature Communications. 2024;15. doi:10.1038/s41467-024-47830-3
Hibshman, G. N., Bravo, J. P. K., Hooper, M. M., Dangerfield, T. L., Zhang, H., Finkelstein, I. J., … Taylor, D. W. (2024). Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-024-47830-3
Hibshman, Grace N., Jack Peter Kelly Bravo, Matthew M. Hooper, Tyler L. Dangerfield, Hongshan Zhang, Ilya J. Finkelstein, Kenneth A. Johnson, and David W. Taylor. “Unraveling the Mechanisms of PAMless DNA Interrogation by SpRY-Cas9.” Nature Communications. Springer Nature, 2024. https://doi.org/10.1038/s41467-024-47830-3.
G. N. Hibshman et al., “Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9,” Nature Communications, vol. 15. Springer Nature, 2024.
Hibshman GN, Bravo JPK, Hooper MM, Dangerfield TL, Zhang H, Finkelstein IJ, Johnson KA, Taylor DW. 2024. Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9. Nature Communications. 15, 3663.
Hibshman, Grace N., et al. “Unraveling the Mechanisms of PAMless DNA Interrogation by SpRY-Cas9.” Nature Communications, vol. 15, 3663, Springer Nature, 2024, doi:10.1038/s41467-024-47830-3.
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