{"external_id":{"pmid":["38688943"]},"article_type":"original","volume":15,"pmid":1,"extern":"1","main_file_link":[{"url":"https://doi.org/10.1038/s41467-024-47830-3","open_access":"1"}],"abstract":[{"text":"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 <jats:italic>Streptococcus pyogenes</jats:italic> 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.","lang":"eng"}],"status":"public","intvolume":"        15","date_updated":"2024-06-04T07:07:38Z","quality_controlled":"1","day":"30","title":"Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9","month":"04","_id":"17113","doi":"10.1038/s41467-024-47830-3","publication_identifier":{"issn":["2041-1723"]},"citation":{"short":"G.N. Hibshman, J.P.K. Bravo, M.M. Hooper, T.L. Dangerfield, H. Zhang, I.J. Finkelstein, K.A. Johnson, D.W. Taylor, Nature Communications 15 (2024).","apa":"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. <i>Nature Communications</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41467-024-47830-3\">https://doi.org/10.1038/s41467-024-47830-3</a>","ama":"Hibshman GN, Bravo JPK, Hooper MM, et al. Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9. <i>Nature Communications</i>. 2024;15. doi:<a href=\"https://doi.org/10.1038/s41467-024-47830-3\">10.1038/s41467-024-47830-3</a>","ista":"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.","mla":"Hibshman, Grace N., et al. “Unraveling the Mechanisms of PAMless DNA Interrogation by SpRY-Cas9.” <i>Nature Communications</i>, vol. 15, 3663, Springer Nature, 2024, doi:<a href=\"https://doi.org/10.1038/s41467-024-47830-3\">10.1038/s41467-024-47830-3</a>.","ieee":"G. N. Hibshman <i>et al.</i>, “Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9,” <i>Nature Communications</i>, vol. 15. Springer Nature, 2024.","chicago":"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.” <i>Nature Communications</i>. Springer Nature, 2024. <a href=\"https://doi.org/10.1038/s41467-024-47830-3\">https://doi.org/10.1038/s41467-024-47830-3</a>."},"type":"journal_article","oa_version":"Published Version","publisher":"Springer Nature","oa":1,"scopus_import":"1","publication_status":"published","author":[{"full_name":"Hibshman, Grace N.","first_name":"Grace N.","last_name":"Hibshman"},{"id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","last_name":"Bravo","full_name":"Bravo, Jack Peter Kelly","first_name":"Jack Peter Kelly","orcid":"0000-0003-0456-0753"},{"last_name":"Hooper","first_name":"Matthew M.","full_name":"Hooper, Matthew M."},{"last_name":"Dangerfield","first_name":"Tyler L.","full_name":"Dangerfield, Tyler L."},{"full_name":"Zhang, Hongshan","first_name":"Hongshan","last_name":"Zhang"},{"last_name":"Finkelstein","first_name":"Ilya J.","full_name":"Finkelstein, Ilya J."},{"first_name":"Kenneth A.","full_name":"Johnson, Kenneth A.","last_name":"Johnson"},{"last_name":"Taylor","full_name":"Taylor, David W.","first_name":"David W."}],"language":[{"iso":"eng"}],"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","date_created":"2024-06-04T06:42:07Z","publication":"Nature Communications","article_number":"3663","article_processing_charge":"Yes","date_published":"2024-04-30T00:00:00Z","year":"2024"}