{"author":[{"id":"91e8ab53-b70a-11ef-adcb-f779f833b451","first_name":"Kaitlyn","last_name":"Kiernan","full_name":"Kiernan, Kaitlyn"},{"full_name":"Taylor, David W.","last_name":"Taylor","first_name":"David W."}],"article_type":"original","file":[{"file_id":"20018","content_type":"application/pdf","file_size":6875712,"file_name":"2025_NatureComm_Kiernan.pdf","access_level":"open_access","success":1,"date_updated":"2025-07-14T08:28:25Z","relation":"main_file","date_created":"2025-07-14T08:28:25Z","creator":"dernst","checksum":"fa9a1eaa7e2e60467768cbaed307aceb"}],"external_id":{"pmid":["40593576"]},"language":[{"iso":"eng"}],"volume":16,"OA_place":"publisher","department":[{"_id":"LeSa"}],"intvolume":"        16","acknowledgement":"We thank Dr. Kenneth Johnson for assistance with kinetic analysis and helpful discussion as well as Dr. Jack Bravo and members of the Taylor lab for insightful comments on the manuscript. Data were collected at the Sauer Structural Biology Laboratory at the University of Texas at Austin. This work was supported by a National Institutes of Health grant R35GM138348 (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. Computational resources for this work were supported by the Welch Foundation grant F-1938 (to D.W.T.).","year":"2025","status":"public","_id":"20002","publication_identifier":{"eissn":["2041-1723"]},"date_updated":"2025-07-14T08:30:06Z","scopus_import":"1","article_number":"5681","PlanS_conform":"1","ddc":["570"],"publication_status":"published","file_date_updated":"2025-07-14T08:28:25Z","quality_controlled":"1","citation":{"chicago":"Kiernan, Kaitlyn, and David W. Taylor. “Visualization of a Multi-Turnover Cas9 after Product Release.” <i>Nature Communications</i>. Springer Nature, 2025. <a href=\"https://doi.org/10.1038/s41467-025-60668-7\">https://doi.org/10.1038/s41467-025-60668-7</a>.","short":"K. Kiernan, D.W. Taylor, Nature Communications 16 (2025).","mla":"Kiernan, Kaitlyn, and David W. Taylor. “Visualization of a Multi-Turnover Cas9 after Product Release.” <i>Nature Communications</i>, vol. 16, 5681, Springer Nature, 2025, doi:<a href=\"https://doi.org/10.1038/s41467-025-60668-7\">10.1038/s41467-025-60668-7</a>.","apa":"Kiernan, K., &#38; Taylor, D. W. (2025). Visualization of a multi-turnover Cas9 after product release. <i>Nature Communications</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41467-025-60668-7\">https://doi.org/10.1038/s41467-025-60668-7</a>","ama":"Kiernan K, Taylor DW. Visualization of a multi-turnover Cas9 after product release. <i>Nature Communications</i>. 2025;16. doi:<a href=\"https://doi.org/10.1038/s41467-025-60668-7\">10.1038/s41467-025-60668-7</a>","ista":"Kiernan K, Taylor DW. 2025. Visualization of a multi-turnover Cas9 after product release. Nature Communications. 16, 5681.","ieee":"K. Kiernan and D. W. Taylor, “Visualization of a multi-turnover Cas9 after product release,” <i>Nature Communications</i>, vol. 16. Springer Nature, 2025."},"OA_type":"gold","abstract":[{"text":"While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Cas9 can eventually be removed from the product by extrinsic factors, such as translocating polymerases, but the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and promote faster enzyme turnover. Using kinetics-guided cryo-EM, we examine the conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.","lang":"eng"}],"doi":"10.1038/s41467-025-60668-7","date_published":"2025-07-01T00:00:00Z","type":"journal_article","tmp":{"short":"CC BY (4.0)","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png"},"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","pmid":1,"oa":1,"DOAJ_listed":"1","date_created":"2025-07-13T22:01:21Z","month":"07","title":"Visualization of a multi-turnover Cas9 after product release","publication":"Nature Communications","day":"01","article_processing_charge":"Yes","publisher":"Springer Nature","oa_version":"Published Version","has_accepted_license":"1"}