[{"quality_controlled":"1","date_published":"2026-01-07T00:00:00Z","publication_identifier":{"eissn":["1476-4687"],"issn":["0028-0836"]},"_id":"20963","department":[{"_id":"JaBr"}],"date_created":"2026-01-08T07:57:17Z","citation":{"apa":"Dmytrenko, O., Yuan, B., Crosby, K. T., Krebel, M., Chen, X., Nowak, J. S., … Beisel, C. L. (2026). RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity. <i>Nature</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41586-025-09852-9\">https://doi.org/10.1038/s41586-025-09852-9</a>","ieee":"O. Dmytrenko <i>et al.</i>, “RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity,” <i>Nature</i>. Springer Nature, 2026.","ama":"Dmytrenko O, Yuan B, Crosby KT, et al. RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity. <i>Nature</i>. 2026. doi:<a href=\"https://doi.org/10.1038/s41586-025-09852-9\">10.1038/s41586-025-09852-9</a>","chicago":"Dmytrenko, Oleg, Biao Yuan, Kadin T. Crosby, Max Krebel, Xiye Chen, Jakub S. Nowak, Andrzej Chramiec-Głąbik, et al. “RNA-Triggered Cas12a3 Cleaves TRNA Tails to Execute Bacterial Immunity.” <i>Nature</i>. Springer Nature, 2026. <a href=\"https://doi.org/10.1038/s41586-025-09852-9\">https://doi.org/10.1038/s41586-025-09852-9</a>.","ista":"Dmytrenko O, Yuan B, Crosby KT, Krebel M, Chen X, Nowak JS, Chramiec-Głąbik A, Filani B, Gribling-Burrer A-S, van der Toorn W, von Kleist M, Achmedov T, Smyth RP, Glatt S, Bravo JPK, Heinz DW, Jackson RN, Beisel CL. 2026. RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity. Nature.","mla":"Dmytrenko, Oleg, et al. “RNA-Triggered Cas12a3 Cleaves TRNA Tails to Execute Bacterial Immunity.” <i>Nature</i>, Springer Nature, 2026, doi:<a href=\"https://doi.org/10.1038/s41586-025-09852-9\">10.1038/s41586-025-09852-9</a>.","short":"O. Dmytrenko, B. Yuan, K.T. Crosby, M. Krebel, X. Chen, J.S. Nowak, A. Chramiec-Głąbik, B. Filani, A.-S. Gribling-Burrer, W. van der Toorn, M. von Kleist, T. Achmedov, R.P. Smyth, S. Glatt, J.P.K. Bravo, D.W. Heinz, R.N. Jackson, C.L. Beisel, Nature (2026)."},"publisher":"Springer Nature","status":"public","month":"01","tmp":{"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","short":"CC BY (4.0)"},"acknowledgement":"We thank Ł. Koziej for processing of the initial cryo-EM datasets, S. Schmelz for support in cryo-EM, A. Gatzemeier for assistance in the purification of dBa1Cas12a3, R. Rarose for support with the in vitro RNA experiments, M. Kaminski for providing purified PsmCas13b protein, L. Schönemann for protein purification, and C. Krempl and S. Backesfor providing the RSV and influenza A transcript-encoding plasmids. This work was supported through funding by the European Research Council (101001394 to S.G.; 865973 and 101158249 to C.L.B.), the R. Gaurth Hansen Family (to R.N.J.), the National Institutes of Health (R35GM138080 to R.N.J.), the PostDoc Plus Program from the Graduate School of Life Sciences at Julius-Maximilians-Universität Würzburg (to O.D.), and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy–The Berlin Mathematics Research Center MATH+ (EXC−2046/1, project ID: 390685689 to M.v.K.). Open access funding provided by Helmholtz-Zentrum für Infektionsforschung GmbH (HZI).","has_accepted_license":"1","OA_place":"publisher","author":[{"first_name":"Oleg","last_name":"Dmytrenko","full_name":"Dmytrenko, Oleg"},{"last_name":"Yuan","first_name":"Biao","full_name":"Yuan, Biao"},{"full_name":"Crosby, Kadin T.","last_name":"Crosby","first_name":"Kadin T."},{"last_name":"Krebel","first_name":"Max","full_name":"Krebel, Max"},{"last_name":"Chen","first_name":"Xiye","full_name":"Chen, Xiye"},{"full_name":"Nowak, Jakub S.","first_name":"Jakub S.","last_name":"Nowak"},{"last_name":"Chramiec-Głąbik","first_name":"Andrzej","full_name":"Chramiec-Głąbik, Andrzej"},{"full_name":"Filani, Bamidele","last_name":"Filani","first_name":"Bamidele"},{"last_name":"Gribling-Burrer","first_name":"Anne-Sophie","full_name":"Gribling-Burrer, Anne-Sophie"},{"full_name":"van der Toorn, Wiep","last_name":"van der Toorn","first_name":"Wiep"},{"first_name":"Max","last_name":"von Kleist","full_name":"von Kleist, Max"},{"last_name":"Achmedov","first_name":"Tatjana","full_name":"Achmedov, Tatjana"},{"full_name":"Smyth, Redmond P.","last_name":"Smyth","first_name":"Redmond P."},{"first_name":"Sebastian","last_name":"Glatt","full_name":"Glatt, Sebastian"},{"last_name":"Bravo","first_name":"Jack Peter Kelly","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","full_name":"Bravo, Jack Peter Kelly","orcid":"0000-0003-0456-0753"},{"full_name":"Heinz, Dirk W.","last_name":"Heinz","first_name":"Dirk W."},{"first_name":"Ryan N.","last_name":"Jackson","full_name":"Jackson, Ryan N."},{"first_name":"Chase L.","last_name":"Beisel","full_name":"Beisel, Chase L."}],"pmid":1,"external_id":{"pmid":["41501459"]},"oa_version":"Published Version","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","PlanS_conform":"1","date_updated":"2026-01-12T10:13:56Z","type":"journal_article","publication":"Nature","ddc":["570"],"scopus_import":"1","article_processing_charge":"Yes (via OA deal)","article_type":"original","OA_type":"hybrid","day":"07","oa":1,"title":"RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity","year":"2026","language":[{"iso":"eng"}],"abstract":[{"text":"In all domains of life, tRNAs mediate the transfer of genetic information from mRNAs to proteins. As their depletion suppresses translation and, consequently, viral replication, tRNAs represent long-standing and increasingly recognized targets of innate immunity1,2,3,4,5. Here we report Cas12a3 effector nucleases from type V CRISPR–Cas adaptive immune systems in bacteria that preferentially cleave tRNAs after recognition of target RNA. Cas12a3 orthologues belong to one of two previously unreported nuclease clades that exhibit RNA-mediated cleavage of non-target RNA, and are distinct from all other known type V systems. Through cell-based and biochemical assays and direct RNA sequencing, we demonstrate that recognition of a complementary target RNA by the CRISPR RNA triggers Cas12a3 to cleave the conserved 5′-CCA-3′ tail of diverse tRNAs to drive growth arrest and anti-phage defence. Cryogenic electron microscopy structures further revealed a distinct tRNA-loading domain that positions the tRNA tail in the RuvC active site of the nuclease. By designing synthetic reporters that mimic the tRNA acceptor stem and tail, we expanded the capacity of current CRISPR-based diagnostics for multiplexed RNA detection. Overall, these findings reveal widespread tRNA inactivation as a previously unrecognized CRISPR-based immune strategy that broadens the application space of the existing CRISPR toolbox.","lang":"eng"}],"main_file_link":[{"url":"https://doi.org/10.1038/s41586-025-09852-9","open_access":"1"}],"doi":"10.1038/s41586-025-09852-9","publication_status":"epub_ahead"},{"month":"10","status":"public","tmp":{"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","short":"CC BY (4.0)"},"page":"5862-5877.e23","department":[{"_id":"JaBr"}],"date_created":"2025-08-07T05:00:04Z","citation":{"ieee":"Z. Zhang <i>et al.</i>, “Kiwa is a membrane-embedded defense supercomplex activated at phage attachment sites,” <i>Cell</i>, vol. 188, no. 21. Elsevier, p. 5862–5877.e23, 2025.","apa":"Zhang, Z., Todeschini, T. C., Wu, Y., Kogay, R., Naji, A., Cardenas Rodriguez, J., … Nobrega, F. L. (2025). Kiwa is a membrane-embedded defense supercomplex activated at phage attachment sites. <i>Cell</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.cell.2025.07.002\">https://doi.org/10.1016/j.cell.2025.07.002</a>","ama":"Zhang Z, Todeschini TC, Wu Y, et al. Kiwa is a membrane-embedded defense supercomplex activated at phage attachment sites. <i>Cell</i>. 2025;188(21):5862-5877.e23. doi:<a href=\"https://doi.org/10.1016/j.cell.2025.07.002\">10.1016/j.cell.2025.07.002</a>","short":"Z. Zhang, T.C. Todeschini, Y. Wu, R. Kogay, A. Naji, J. Cardenas Rodriguez, R. Mondi, D. Kaganovich, D.W. Taylor, J.P.K. Bravo, M. Teplova, T. Amen, E. Koonin, D.J. Patel, F.L. Nobrega, Cell 188 (2025) 5862–5877.e23.","mla":"Zhang, Zhiying, et al. “Kiwa Is a Membrane-Embedded Defense Supercomplex Activated at Phage Attachment Sites.” <i>Cell</i>, vol. 188, no. 21, Elsevier, 2025, p. 5862–5877.e23, doi:<a href=\"https://doi.org/10.1016/j.cell.2025.07.002\">10.1016/j.cell.2025.07.002</a>.","ista":"Zhang Z, Todeschini TC, Wu Y, Kogay R, Naji A, Cardenas Rodriguez J, Mondi R, Kaganovich D, Taylor DW, Bravo JPK, Teplova M, Amen T, Koonin E, Patel DJ, Nobrega FL. 2025. Kiwa is a membrane-embedded defense supercomplex activated at phage attachment sites. Cell. 188(21), 5862–5877.e23.","chicago":"Zhang, Zhiying, Thomas C. Todeschini, Yi Wu, Roman Kogay, Ameena Naji, Joaquin Cardenas Rodriguez, Rupavidhya Mondi, et al. “Kiwa Is a Membrane-Embedded Defense Supercomplex Activated at Phage Attachment Sites.” <i>Cell</i>. Elsevier, 2025. <a href=\"https://doi.org/10.1016/j.cell.2025.07.002\">https://doi.org/10.1016/j.cell.2025.07.002</a>."},"publisher":"Elsevier","_id":"20143","publication_identifier":{"issn":["0092-8674"],"eissn":["1097-4172"]},"issue":"21","intvolume":"       188","quality_controlled":"1","date_published":"2025-10-16T00:00:00Z","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","oa_version":"Published Version","isi":1,"OA_place":"publisher","external_id":{"pmid":["40730155"],"isi":["001603560700005"]},"author":[{"first_name":"Zhiying","last_name":"Zhang","full_name":"Zhang, Zhiying"},{"full_name":"Todeschini, Thomas C.","last_name":"Todeschini","first_name":"Thomas C."},{"last_name":"Wu","first_name":"Yi","full_name":"Wu, Yi"},{"full_name":"Kogay, Roman","first_name":"Roman","last_name":"Kogay"},{"first_name":"Ameena","last_name":"Naji","full_name":"Naji, Ameena"},{"first_name":"Joaquin","last_name":"Cardenas Rodriguez","full_name":"Cardenas Rodriguez, Joaquin"},{"full_name":"Mondi, Rupavidhya","first_name":"Rupavidhya","last_name":"Mondi"},{"full_name":"Kaganovich, Daniel","last_name":"Kaganovich","first_name":"Daniel"},{"full_name":"Taylor, David W.","last_name":"Taylor","first_name":"David W."},{"id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753","full_name":"Bravo, Jack Peter Kelly","first_name":"Jack Peter Kelly","last_name":"Bravo"},{"full_name":"Teplova, Marianna","first_name":"Marianna","last_name":"Teplova"},{"first_name":"Triana","last_name":"Amen","full_name":"Amen, Triana"},{"first_name":"Eugene","last_name":"Koonin","full_name":"Koonin, Eugene"},{"full_name":"Patel, Dinshaw J.","last_name":"Patel","first_name":"Dinshaw J."},{"full_name":"Nobrega, Franklin L.","last_name":"Nobrega","first_name":"Franklin L."}],"pmid":1,"file":[{"content_type":"application/pdf","creator":"dernst","date_updated":"2025-12-29T14:15:25Z","file_id":"20875","file_name":"2025_Cell_Zhang.pdf","checksum":"b944de5fbd7455f58e1ff338ad352239","success":1,"file_size":32104588,"access_level":"open_access","relation":"main_file","date_created":"2025-12-29T14:15:25Z"}],"acknowledgement":"We thank Rotem Sorek (Weizmann Institute of Science) for the Lambda Gam mutant and Ian Molineux (University of Texas) for T4Δgp2. We thank You Yu (Zhejiang University-University of Edinburgh Institute) and J. De La Cruz (MSK) for assistance with cryo-EM data collection and Lyuqin Zheng (MSK) for discussions on structural analysis. We thank the Imaging and Microscopy Centre (IMC) at the University of Southampton. This work was supported by Royal Society grant RGS\\R2\\222312 to F.L.N.; Welch Foundation grant F-1938 and National Institutes of Health R35GM138348 to D.W.T.; Wessex Medical Research Innovation grant AE06 to T.A.; and NIH grant GM145888 and Maloris Foundation and Memorial Sloan-Kettering Core grant (P30-CA008748) to D.J.P. In addition to MSKCC cryo-EM resources, some of this work was performed at the National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129539) and Simons Foundation (SF349247) and NY State Assembly grants. This research used NSLS-II MX X-ray User Resources (FMX) of the National Synchrotron Light Source II, operated for the DOE Office of Science by Brookhaven National Laboratory under contract no. DE-SC0012704. The Center for BioMolecular Structure (CBMS) is primarily supported by the NIH, the National Institute of General Medical Sciences (NIGMS) through a Center Core P30 Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1605010). R.K. and E.V.K. are supported by the Intramural Research Program of the NIH (National Library of Medicine).","has_accepted_license":"1","article_processing_charge":"Yes (in subscription journal)","article_type":"original","scopus_import":"1","ddc":["570"],"publication":"Cell","type":"journal_article","PlanS_conform":"1","date_updated":"2025-12-29T14:15:58Z","doi":"10.1016/j.cell.2025.07.002","publication_status":"published","file_date_updated":"2025-12-29T14:15:25Z","language":[{"iso":"eng"}],"abstract":[{"lang":"eng","text":"Bacteria and archaea deploy diverse antiviral defense systems, many of which remain mechanistically uncharacterized. Here, we characterize Kiwa, a widespread two-component system composed of the transmembrane sensor KwaA and the DNA-binding effector KwaB. Cryogenic electron microscopy (cryo-EM) analysis reveals that KwaA and KwaB assemble into a large, membrane-associated supercomplex. Upon phage binding, KwaA senses infection at the membrane, leading to KwaB binding of ejected phage DNA and inhibition of replication and late transcription, without inducing host cell death. Although KwaB can bind DNA independently, its antiviral activity requires association with KwaA, suggesting spatial or conformational regulation. We show that the phage-encoded DNA-mimic protein Gam directly binds and inhibits KwaB but that co-expression with the Gam-targeted RecBCD system restores protection by Kiwa. Our findings support a model in which Kiwa coordinates membrane-associated detection of phage infection with downstream DNA binding by its effector, forming a spatially coordinated antiviral mechanism."}],"oa":1,"OA_type":"hybrid","day":"16","year":"2025","volume":188,"title":"Kiwa is a membrane-embedded defense supercomplex activated at phage attachment sites"},{"ddc":["570"],"scopus_import":"1","date_updated":"2025-07-03T11:58:22Z","type":"journal_article","publication":"Nature Communications","article_type":"original","article_processing_charge":"Yes","abstract":[{"lang":"eng","text":"Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains for DNA recognition. The sgRNA can be truncated by up to ~25% yet still retain activity in vivo. Using ancestral sequence reconstruction, we generated compact nucleases capable of efficient genome editing in mammalian cells. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for future re-engineering of minimal RNA-guided DNA endonucleases."}],"article_number":"457","language":[{"iso":"eng"}],"volume":16,"title":"DNA targeting by compact Cas9d and its resurrected ancestor","year":"2025","OA_type":"gold","day":"07","oa":1,"publication_status":"published","doi":"10.1038/s41467-024-55573-4","file_date_updated":"2025-01-22T14:35:22Z","publication_identifier":{"eissn":["2041-1723"]},"intvolume":"        16","_id":"18848","date_published":"2025-01-07T00:00:00Z","quality_controlled":"1","tmp":{"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","short":"CC BY (4.0)"},"status":"public","month":"01","citation":{"apa":"Ocampo, R. F., Bravo, J. P. K., Dangerfield, T. L., Nocedal, I., Jirde, S. A., Alexander, L. M., … Taylor, D. W. (2025). DNA targeting by compact Cas9d and its resurrected ancestor. <i>Nature Communications</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41467-024-55573-4\">https://doi.org/10.1038/s41467-024-55573-4</a>","ieee":"R. F. Ocampo <i>et al.</i>, “DNA targeting by compact Cas9d and its resurrected ancestor,” <i>Nature Communications</i>, vol. 16. Springer Nature, 2025.","ama":"Ocampo RF, Bravo JPK, Dangerfield TL, et al. DNA targeting by compact Cas9d and its resurrected ancestor. <i>Nature Communications</i>. 2025;16. doi:<a href=\"https://doi.org/10.1038/s41467-024-55573-4\">10.1038/s41467-024-55573-4</a>","chicago":"Ocampo, Rodrigo Fregoso, Jack Peter Kelly Bravo, Tyler L. Dangerfield, Isabel Nocedal, Samatar A. Jirde, Lisa M. Alexander, Nicole C. Thomas, et al. “DNA Targeting by Compact Cas9d and Its Resurrected Ancestor.” <i>Nature Communications</i>. Springer Nature, 2025. <a href=\"https://doi.org/10.1038/s41467-024-55573-4\">https://doi.org/10.1038/s41467-024-55573-4</a>.","mla":"Ocampo, Rodrigo Fregoso, et al. “DNA Targeting by Compact Cas9d and Its Resurrected Ancestor.” <i>Nature Communications</i>, vol. 16, 457, Springer Nature, 2025, doi:<a href=\"https://doi.org/10.1038/s41467-024-55573-4\">10.1038/s41467-024-55573-4</a>.","short":"R.F. Ocampo, J.P.K. Bravo, T.L. Dangerfield, I. Nocedal, S.A. Jirde, L.M. Alexander, N.C. Thomas, A. Das, S. Nielson, K.A. Johnson, C.T. Brown, C.N. Butterfield, D.S.A. Goltsman, D.W. Taylor, Nature Communications 16 (2025).","ista":"Ocampo RF, Bravo JPK, Dangerfield TL, Nocedal I, Jirde SA, Alexander LM, Thomas NC, Das A, Nielson S, Johnson KA, Brown CT, Butterfield CN, Goltsman DSA, Taylor DW. 2025. DNA targeting by compact Cas9d and its resurrected ancestor. Nature Communications. 16, 457."},"publisher":"Springer Nature","department":[{"_id":"JaBr"}],"date_created":"2025-01-19T23:01:50Z","acknowledgement":"We would like to thank M. Ocampo Camacho and M.F. Canedo Ocampo for assistance with the figures. We thank M. Hooper for assistance developing the GFP assay and operating the CE machine for in vitro cleavage analysis. We thank E. Schwartz and A. Brilot for expert cryo-EM support in the Sauer Structural Biology Laboratory at UT Austin. This work was funded, in part, by a sponsored research agreement with Metagenomi, Inc. (to D.W.T), a Welch Foundation Research Grant F-1938 (to D.W.T), and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation Medical Research Grant (to D.W.T), and a grant from the National Institute of Allergy and Infectious Diseases (NIAID 1R01AI110577 to K.A.J.).","has_accepted_license":"1","file":[{"file_id":"18869","date_updated":"2025-01-22T14:35:22Z","creator":"dernst","content_type":"application/pdf","date_created":"2025-01-22T14:35:22Z","relation":"main_file","access_level":"open_access","success":1,"checksum":"885e96690620790d5c9f188a1587b4cd","file_size":5450660,"file_name":"2025_NatureComm_Ocampo.pdf"}],"oa_version":"Published Version","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","pmid":1,"author":[{"full_name":"Ocampo, Rodrigo Fregoso","last_name":"Ocampo","first_name":"Rodrigo Fregoso"},{"last_name":"Bravo","first_name":"Jack Peter Kelly","orcid":"0000-0003-0456-0753","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","full_name":"Bravo, Jack Peter Kelly"},{"first_name":"Tyler L.","last_name":"Dangerfield","full_name":"Dangerfield, Tyler L."},{"full_name":"Nocedal, Isabel","last_name":"Nocedal","first_name":"Isabel"},{"last_name":"Jirde","first_name":"Samatar A.","full_name":"Jirde, Samatar A."},{"last_name":"Alexander","first_name":"Lisa M.","full_name":"Alexander, Lisa M."},{"last_name":"Thomas","first_name":"Nicole C.","full_name":"Thomas, Nicole C."},{"last_name":"Das","first_name":"Anjali","full_name":"Das, Anjali"},{"first_name":"Sarah","last_name":"Nielson","full_name":"Nielson, Sarah"},{"first_name":"Kenneth A.","last_name":"Johnson","full_name":"Johnson, Kenneth A."},{"first_name":"Christopher T.","last_name":"Brown","full_name":"Brown, Christopher T."},{"first_name":"Cristina N.","last_name":"Butterfield","full_name":"Butterfield, Cristina N."},{"full_name":"Goltsman, Daniela S.A.","first_name":"Daniela S.A.","last_name":"Goltsman"},{"last_name":"Taylor","first_name":"David W.","full_name":"Taylor, David W."}],"DOAJ_listed":"1","external_id":{"pmid":["39774105"]},"OA_place":"publisher"},{"oa":1,"day":"30","year":"2024","volume":15,"title":"Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9","article_number":"3663","language":[{"iso":"eng"}],"abstract":[{"lang":"eng","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 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."}],"corr_author":"1","file_date_updated":"2024-05-13T11:46:19Z","doi":"10.1038/s41467-024-47830-3","publication_status":"published","publication":"Nature Communications","type":"journal_article","date_updated":"2025-05-14T09:33:21Z","scopus_import":"1","ddc":["570"],"article_processing_charge":"Yes","article_type":"original","file":[{"file_size":7477013,"checksum":"509c65919067a03ef8ad65c7192cd860","success":1,"file_name":"2024_NatureComm_Hibshman.pdf","date_created":"2024-05-13T11:46:19Z","relation":"main_file","access_level":"open_access","creator":"dernst","content_type":"application/pdf","date_updated":"2024-05-13T11:46:19Z","file_id":"15386"}],"has_accepted_license":"1","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.","external_id":{"pmid":["38688943"]},"DOAJ_listed":"1","pmid":1,"author":[{"last_name":"Hibshman","first_name":"Grace N.","full_name":"Hibshman, Grace N."},{"last_name":"Bravo","first_name":"Jack Peter Kelly","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753","full_name":"Bravo, Jack Peter Kelly"},{"first_name":"Matthew M.","last_name":"Hooper","full_name":"Hooper, Matthew M."},{"full_name":"Dangerfield, Tyler L.","last_name":"Dangerfield","first_name":"Tyler L."},{"first_name":"Hongshan","last_name":"Zhang","full_name":"Zhang, Hongshan"},{"first_name":"Ilya J.","last_name":"Finkelstein","full_name":"Finkelstein, Ilya J."},{"last_name":"Johnson","first_name":"Kenneth A.","full_name":"Johnson, Kenneth A."},{"full_name":"Taylor, David W.","first_name":"David W.","last_name":"Taylor"}],"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","oa_version":"Published Version","quality_controlled":"1","date_published":"2024-04-30T00:00:00Z","_id":"15372","intvolume":"        15","publication_identifier":{"eissn":["2041-1723"]},"date_created":"2024-05-12T22:01:00Z","department":[{"_id":"JaBr"}],"publisher":"Springer Nature","citation":{"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>","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.","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>","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>.","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>.","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)."},"month":"04","status":"public","tmp":{"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","short":"CC BY (4.0)"}},{"article_processing_charge":"No","article_type":"original","date_updated":"2025-06-24T12:47:21Z","type":"journal_article","publication":"Nature","scopus_import":"1","main_file_link":[{"url":"https://pmc.ncbi.nlm.nih.gov/articles/PMC11649018/","open_access":"1"}],"doi":"10.1038/s41586-024-07515-9","publication_status":"published","day":"27","OA_type":"green","oa":1,"volume":630,"title":"Plasmid targeting and destruction by the DdmDE bacterial defence system","year":"2024","language":[{"iso":"eng"}],"abstract":[{"text":"Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE–guide–target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.\r\n\r\n","lang":"eng"}],"corr_author":"1","date_created":"2024-08-19T09:41:18Z","department":[{"_id":"JaBr"}],"publisher":"Springer Nature","citation":{"chicago":"Bravo, Jack Peter Kelly, Delisa A. Ramos, Rodrigo Fregoso Ocampo, Caiden Ingram, and David W. Taylor. “Plasmid Targeting and Destruction by the DdmDE Bacterial Defence System.” <i>Nature</i>. Springer Nature, 2024. <a href=\"https://doi.org/10.1038/s41586-024-07515-9\">https://doi.org/10.1038/s41586-024-07515-9</a>.","ista":"Bravo JPK, Ramos DA, Fregoso Ocampo R, Ingram C, Taylor DW. 2024. Plasmid targeting and destruction by the DdmDE bacterial defence system. Nature. 630(8018), 961–967.","short":"J.P.K. Bravo, D.A. Ramos, R. Fregoso Ocampo, C. Ingram, D.W. Taylor, Nature 630 (2024) 961–967.","mla":"Bravo, Jack Peter Kelly, et al. “Plasmid Targeting and Destruction by the DdmDE Bacterial Defence System.” <i>Nature</i>, vol. 630, no. 8018, Springer Nature, 2024, pp. 961–67, doi:<a href=\"https://doi.org/10.1038/s41586-024-07515-9\">10.1038/s41586-024-07515-9</a>.","apa":"Bravo, J. P. K., Ramos, D. A., Fregoso Ocampo, R., Ingram, C., &#38; Taylor, D. W. (2024). Plasmid targeting and destruction by the DdmDE bacterial defence system. <i>Nature</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41586-024-07515-9\">https://doi.org/10.1038/s41586-024-07515-9</a>","ama":"Bravo JPK, Ramos DA, Fregoso Ocampo R, Ingram C, Taylor DW. Plasmid targeting and destruction by the DdmDE bacterial defence system. <i>Nature</i>. 2024;630(8018):961-967. doi:<a href=\"https://doi.org/10.1038/s41586-024-07515-9\">10.1038/s41586-024-07515-9</a>","ieee":"J. P. K. Bravo, D. A. Ramos, R. Fregoso Ocampo, C. Ingram, and D. W. Taylor, “Plasmid targeting and destruction by the DdmDE bacterial defence system,” <i>Nature</i>, vol. 630, no. 8018. Springer Nature, pp. 961–967, 2024."},"status":"public","month":"06","page":"961-967","quality_controlled":"1","date_published":"2024-06-27T00:00:00Z","intvolume":"       630","issue":"8018","publication_identifier":{"issn":["0028-0836"],"eissn":["1476-4687"]},"_id":"17442","OA_place":"repository","pmid":1,"author":[{"orcid":"0000-0003-0456-0753","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","full_name":"Bravo, Jack Peter Kelly","last_name":"Bravo","first_name":"Jack Peter Kelly"},{"last_name":"Ramos","first_name":"Delisa A.","full_name":"Ramos, Delisa A."},{"full_name":"Fregoso Ocampo, Rodrigo","last_name":"Fregoso Ocampo","first_name":"Rodrigo"},{"first_name":"Caiden","last_name":"Ingram","full_name":"Ingram, Caiden"},{"last_name":"Taylor","first_name":"David W.","full_name":"Taylor, David W."}],"external_id":{"pmid":["38740055"]},"oa_version":"Submitted Version","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","acknowledgement":"We thank K. Kiernan, G. Hibshman and I. Strohkendl for insightful discussions and comments on the manuscript, and R. Lin for assistance with the ATPase assay. Data were collected at the Sauer Structural Biology Laboratory at the University of Texas at Austin. This work was supported in part by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) R35GM138348 (to D.W.T.) and Welch Foundation research grant F-1938 (to D.W.T.)."},{"doi":"10.1080/17460913.2024.2389720","publication_status":"published","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1080/17460913.2024.2389720"}],"language":[{"iso":"eng"}],"corr_author":"1","OA_type":"free access","day":"01","oa":1,"title":"Anti-plasmid immunity: A key to pathogen success?","volume":19,"year":"2024","article_processing_charge":"No","article_type":"letter_note","scopus_import":"1","date_updated":"2025-09-08T09:03:00Z","type":"journal_article","publication":"Future Microbiology","oa_version":"Published Version","user_id":"317138e5-6ab7-11ef-aa6d-ffef3953e345","OA_place":"publisher","isi":1,"author":[{"id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753","full_name":"Bravo, Jack Peter Kelly","first_name":"Jack Peter Kelly","last_name":"Bravo"}],"pmid":1,"external_id":{"pmid":["39230568"],"isi":["001306115400001"]},"acknowledgement":"I would like to thank K Kiernan for insightful comments and feedback. J P K Bravo is supported by IST Austria.","has_accepted_license":"1","status":"public","month":"10","page":"1269-1272","department":[{"_id":"JaBr"}],"date_created":"2024-09-05T07:32:00Z","publisher":"Taylor & Francis","citation":{"ieee":"J. P. K. Bravo, “Anti-plasmid immunity: A key to pathogen success?,” <i>Future Microbiology</i>, vol. 19, no. 15. Taylor &#38; Francis, pp. 1269–1272, 2024.","ama":"Bravo JPK. Anti-plasmid immunity: A key to pathogen success? <i>Future Microbiology</i>. 2024;19(15):1269-1272. doi:<a href=\"https://doi.org/10.1080/17460913.2024.2389720\">10.1080/17460913.2024.2389720</a>","apa":"Bravo, J. P. K. (2024). Anti-plasmid immunity: A key to pathogen success? <i>Future Microbiology</i>. Taylor &#38; Francis. <a href=\"https://doi.org/10.1080/17460913.2024.2389720\">https://doi.org/10.1080/17460913.2024.2389720</a>","mla":"Bravo, Jack Peter Kelly. “Anti-Plasmid Immunity: A Key to Pathogen Success?” <i>Future Microbiology</i>, vol. 19, no. 15, Taylor &#38; Francis, 2024, pp. 1269–72, doi:<a href=\"https://doi.org/10.1080/17460913.2024.2389720\">10.1080/17460913.2024.2389720</a>.","short":"J.P.K. Bravo, Future Microbiology 19 (2024) 1269–1272.","ista":"Bravo JPK. 2024. Anti-plasmid immunity: A key to pathogen success? Future Microbiology. 19(15), 1269–1272.","chicago":"Bravo, Jack Peter Kelly. “Anti-Plasmid Immunity: A Key to Pathogen Success?” <i>Future Microbiology</i>. Taylor &#38; Francis, 2024. <a href=\"https://doi.org/10.1080/17460913.2024.2389720\">https://doi.org/10.1080/17460913.2024.2389720</a>."},"intvolume":"        19","issue":"15","publication_identifier":{"eissn":["1746-0921"],"issn":["1746-0913"]},"_id":"17494","quality_controlled":"1","date_published":"2024-10-01T00:00:00Z"}]