[{"date_updated":"2026-01-12T10:13:56Z","year":"2026","author":[{"full_name":"Dmytrenko, Oleg","first_name":"Oleg","last_name":"Dmytrenko"},{"full_name":"Yuan, Biao","first_name":"Biao","last_name":"Yuan"},{"last_name":"Crosby","first_name":"Kadin T.","full_name":"Crosby, Kadin T."},{"full_name":"Krebel, Max","last_name":"Krebel","first_name":"Max"},{"first_name":"Xiye","last_name":"Chen","full_name":"Chen, Xiye"},{"first_name":"Jakub S.","last_name":"Nowak","full_name":"Nowak, Jakub S."},{"last_name":"Chramiec-Głąbik","first_name":"Andrzej","full_name":"Chramiec-Głąbik, Andrzej"},{"first_name":"Bamidele","last_name":"Filani","full_name":"Filani, 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"},{"last_name":"von Kleist","first_name":"Max","full_name":"von Kleist, Max"},{"full_name":"Achmedov, Tatjana","first_name":"Tatjana","last_name":"Achmedov"},{"first_name":"Redmond P.","last_name":"Smyth","full_name":"Smyth, Redmond P."},{"first_name":"Sebastian","last_name":"Glatt","full_name":"Glatt, Sebastian"},{"full_name":"Bravo, Jack Peter Kelly","first_name":"Jack Peter Kelly","last_name":"Bravo","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753"},{"full_name":"Heinz, Dirk W.","first_name":"Dirk W.","last_name":"Heinz"},{"first_name":"Ryan N.","last_name":"Jackson","full_name":"Jackson, Ryan N."},{"full_name":"Beisel, Chase L.","first_name":"Chase L.","last_name":"Beisel"}],"publication":"Nature","status":"public","OA_type":"hybrid","ddc":["570"],"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>","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>","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>.","ieee":"O. Dmytrenko <i>et al.</i>, “RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity,” <i>Nature</i>. Springer Nature, 2026.","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.","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).","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>."},"scopus_import":"1","has_accepted_license":"1","publisher":"Springer Nature","PlanS_conform":"1","day":"07","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","OA_place":"publisher","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"quality_controlled":"1","article_type":"original","oa":1,"date_published":"2026-01-07T00:00:00Z","_id":"20963","publication_status":"epub_ahead","pmid":1,"language":[{"iso":"eng"}],"title":"RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity","article_processing_charge":"Yes (via OA deal)","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1038/s41586-025-09852-9"}],"date_created":"2026-01-08T07:57:17Z","license":"https://creativecommons.org/licenses/by/4.0/","doi":"10.1038/s41586-025-09852-9","oa_version":"Published Version","department":[{"_id":"JaBr"}],"publication_identifier":{"eissn":["1476-4687"],"issn":["0028-0836"]},"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).","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"}],"month":"01","type":"journal_article","external_id":{"pmid":["41501459"]}},{"publisher":"Elsevier","file":[{"date_updated":"2025-12-29T14:15:25Z","access_level":"open_access","date_created":"2025-12-29T14:15:25Z","success":1,"file_size":32104588,"content_type":"application/pdf","checksum":"b944de5fbd7455f58e1ff338ad352239","creator":"dernst","file_name":"2025_Cell_Zhang.pdf","relation":"main_file","file_id":"20875"}],"has_accepted_license":"1","scopus_import":"1","OA_type":"hybrid","citation":{"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>.","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>","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.","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.","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>."},"ddc":["570"],"page":"5862-5877.e23","status":"public","publication":"Cell","author":[{"full_name":"Zhang, Zhiying","first_name":"Zhiying","last_name":"Zhang"},{"full_name":"Todeschini, Thomas C.","first_name":"Thomas C.","last_name":"Todeschini"},{"full_name":"Wu, Yi","last_name":"Wu","first_name":"Yi"},{"last_name":"Kogay","first_name":"Roman","full_name":"Kogay, Roman"},{"full_name":"Naji, Ameena","last_name":"Naji","first_name":"Ameena"},{"full_name":"Cardenas Rodriguez, Joaquin","first_name":"Joaquin","last_name":"Cardenas Rodriguez"},{"first_name":"Rupavidhya","last_name":"Mondi","full_name":"Mondi, Rupavidhya"},{"last_name":"Kaganovich","first_name":"Daniel","full_name":"Kaganovich, Daniel"},{"full_name":"Taylor, David W.","last_name":"Taylor","first_name":"David W."},{"full_name":"Bravo, Jack Peter Kelly","first_name":"Jack Peter Kelly","last_name":"Bravo","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753"},{"full_name":"Teplova, Marianna","last_name":"Teplova","first_name":"Marianna"},{"last_name":"Amen","first_name":"Triana","full_name":"Amen, Triana"},{"first_name":"Eugene","last_name":"Koonin","full_name":"Koonin, Eugene"},{"first_name":"Dinshaw J.","last_name":"Patel","full_name":"Patel, Dinshaw J."},{"full_name":"Nobrega, Franklin L.","first_name":"Franklin L.","last_name":"Nobrega"}],"year":"2025","date_updated":"2025-12-29T14:15:58Z","oa":1,"article_type":"original","quality_controlled":"1","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"OA_place":"publisher","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","day":"16","issue":"21","PlanS_conform":"1","volume":188,"language":[{"iso":"eng"}],"pmid":1,"intvolume":"       188","publication_status":"published","_id":"20143","isi":1,"date_published":"2025-10-16T00:00:00Z","external_id":{"pmid":["40730155"],"isi":["001603560700005"]},"file_date_updated":"2025-12-29T14:15:25Z","type":"journal_article","month":"10","abstract":[{"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.","lang":"eng"}],"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).","publication_identifier":{"eissn":["1097-4172"],"issn":["0092-8674"]},"department":[{"_id":"JaBr"}],"oa_version":"Published Version","doi":"10.1016/j.cell.2025.07.002","date_created":"2025-08-07T05:00:04Z","article_processing_charge":"Yes (in subscription journal)","title":"Kiwa is a membrane-embedded defense supercomplex activated at phage attachment sites"},{"article_processing_charge":"Yes","oa_version":"Published Version","doi":"10.1038/s41467-024-55573-4","date_created":"2025-01-19T23:01:50Z","title":"DNA targeting by compact Cas9d and its resurrected ancestor","department":[{"_id":"JaBr"}],"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.).","publication_identifier":{"eissn":["2041-1723"]},"external_id":{"pmid":["39774105"]},"abstract":[{"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.","lang":"eng"}],"type":"journal_article","file_date_updated":"2025-01-22T14:35:22Z","month":"01","date_published":"2025-01-07T00:00:00Z","_id":"18848","pmid":1,"DOAJ_listed":"1","publication_status":"published","intvolume":"        16","language":[{"iso":"eng"}],"volume":16,"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","OA_place":"publisher","day":"07","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"oa":1,"quality_controlled":"1","article_type":"original","date_updated":"2025-07-03T11:58:22Z","year":"2025","author":[{"first_name":"Rodrigo Fregoso","last_name":"Ocampo","full_name":"Ocampo, Rodrigo Fregoso"},{"full_name":"Bravo, Jack Peter Kelly","first_name":"Jack Peter Kelly","last_name":"Bravo","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753"},{"last_name":"Dangerfield","first_name":"Tyler L.","full_name":"Dangerfield, Tyler L."},{"last_name":"Nocedal","first_name":"Isabel","full_name":"Nocedal, Isabel"},{"full_name":"Jirde, Samatar A.","last_name":"Jirde","first_name":"Samatar A."},{"full_name":"Alexander, Lisa M.","first_name":"Lisa M.","last_name":"Alexander"},{"first_name":"Nicole C.","last_name":"Thomas","full_name":"Thomas, Nicole C."},{"full_name":"Das, Anjali","last_name":"Das","first_name":"Anjali"},{"last_name":"Nielson","first_name":"Sarah","full_name":"Nielson, Sarah"},{"first_name":"Kenneth A.","last_name":"Johnson","full_name":"Johnson, Kenneth A."},{"last_name":"Brown","first_name":"Christopher T.","full_name":"Brown, Christopher T."},{"full_name":"Butterfield, Cristina N.","last_name":"Butterfield","first_name":"Cristina N."},{"first_name":"Daniela S.A.","last_name":"Goltsman","full_name":"Goltsman, Daniela S.A."},{"last_name":"Taylor","first_name":"David W.","full_name":"Taylor, David W."}],"OA_type":"gold","ddc":["570"],"article_number":"457","citation":{"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).","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>.","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.","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>.","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>","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.","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>"},"publication":"Nature Communications","status":"public","scopus_import":"1","has_accepted_license":"1","file":[{"success":1,"date_created":"2025-01-22T14:35:22Z","date_updated":"2025-01-22T14:35:22Z","access_level":"open_access","content_type":"application/pdf","checksum":"885e96690620790d5c9f188a1587b4cd","file_size":5450660,"relation":"main_file","file_id":"18869","creator":"dernst","file_name":"2025_NatureComm_Ocampo.pdf"}],"publisher":"Springer Nature"},{"publication_identifier":{"issn":["2041-1723"]},"type":"journal_article","file_date_updated":"2024-11-12T10:18:32Z","month":"11","abstract":[{"text":"Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues. Here, we sought to redesign PsCas9 for in vivo editing using lipid nanoparticles. We solve the PsCas9 ribonucleoprotein structure with cryo-EM and characterize it biochemically, providing a basis for its rational engineering. Screening over numerous guide RNA and protein variants lead us to develop engineered PsCas9 (ePsCas9) with up to 20-fold increased activity across various targets and preserved safety advantages. We apply the same design principles to boost the activity of FnCas9, an enzyme phylogenetically relevant to PsCas9. Remarkably, a single administration of mRNA encoding ePsCas9 and its guide formulated with lipid nanoparticles results in high levels of editing in the Pcsk9 gene in mouse liver, a clinically relevant target for hypercholesterolemia treatment. Collectively, our findings introduce ePsCas9 as a highly efficient, and precise tool for therapeutic genome editing, in addition to the engineering strategy applicable to other Cas9 orthologues.","lang":"eng"}],"title":"Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles","oa_version":"Published Version","license":"https://creativecommons.org/licenses/by-nc-nd/4.0/","doi":"10.1038/s41467-024-53418-8","date_created":"2024-11-12T10:18:04Z","article_processing_charge":"Yes","intvolume":"        15","DOAJ_listed":"1","publication_status":"published","language":[{"iso":"eng"}],"date_published":"2024-11-07T00:00:00Z","_id":"18545","tmp":{"name":"Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)","short":"CC BY-NC-ND (4.0)","image":"/images/cc_by_nc_nd.png","legal_code_url":"https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode"},"article_type":"original","quality_controlled":"1","oa":1,"volume":15,"day":"07","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","OA_place":"publisher","status":"public","publication":"Nature Communications","ddc":["572"],"OA_type":"gold","article_number":"9173","citation":{"short":"D. Degtev, J.P.K. Bravo, A. Emmanouilidi, A. Zdravković, O.K. Choong, J. Liz Touza, N. Selfjord, I. Weisheit, M. Francescatto, P. Akcakaya, M. Porritt, M. Maresca, D. Taylor, G. Sienski, Nature Communications 15 (2024).","chicago":"Degtev, Dmitrii, Jack Peter Kelly Bravo, Aikaterini Emmanouilidi, Aleksandar Zdravković, Oi Kuan Choong, Julia Liz Touza, Niklas Selfjord, et al. “Engineered PsCas9 Enables Therapeutic Genome Editing in Mouse Liver with Lipid Nanoparticles.” <i>Nature Communications</i>. Springer Nature, 2024. <a href=\"https://doi.org/10.1038/s41467-024-53418-8\">https://doi.org/10.1038/s41467-024-53418-8</a>.","ista":"Degtev D, Bravo JPK, Emmanouilidi A, Zdravković A, Choong OK, Liz Touza J, Selfjord N, Weisheit I, Francescatto M, Akcakaya P, Porritt M, Maresca M, Taylor D, Sienski G. 2024. Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles. Nature Communications. 15, 9173.","apa":"Degtev, D., Bravo, J. P. K., Emmanouilidi, A., Zdravković, A., Choong, O. K., Liz Touza, J., … Sienski, G. (2024). Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles. <i>Nature Communications</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41467-024-53418-8\">https://doi.org/10.1038/s41467-024-53418-8</a>","mla":"Degtev, Dmitrii, et al. “Engineered PsCas9 Enables Therapeutic Genome Editing in Mouse Liver with Lipid Nanoparticles.” <i>Nature Communications</i>, vol. 15, 9173, Springer Nature, 2024, doi:<a href=\"https://doi.org/10.1038/s41467-024-53418-8\">10.1038/s41467-024-53418-8</a>.","ama":"Degtev D, Bravo JPK, Emmanouilidi A, et al. Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles. <i>Nature Communications</i>. 2024;15. doi:<a href=\"https://doi.org/10.1038/s41467-024-53418-8\">10.1038/s41467-024-53418-8</a>","ieee":"D. Degtev <i>et al.</i>, “Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles,” <i>Nature Communications</i>, vol. 15. Springer Nature, 2024."},"file":[{"creator":"jbravo","file_name":"s41467-024-53418-8.pdf","relation":"main_file","file_id":"18546","access_level":"open_access","date_updated":"2024-11-12T10:18:32Z","success":1,"date_created":"2024-11-12T10:18:32Z","content_type":"application/pdf","file_size":2967001,"checksum":"dcfadc806f4144d065eb8e2032554782"}],"publisher":"Springer Nature","has_accepted_license":"1","scopus_import":"1","date_updated":"2024-11-13T08:19:50Z","extern":"1","author":[{"last_name":"Degtev","first_name":"Dmitrii","full_name":"Degtev, Dmitrii"},{"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"},{"first_name":"Aikaterini","last_name":"Emmanouilidi","full_name":"Emmanouilidi, Aikaterini"},{"first_name":"Aleksandar","last_name":"Zdravković","full_name":"Zdravković, Aleksandar"},{"full_name":"Choong, Oi Kuan","first_name":"Oi Kuan","last_name":"Choong"},{"full_name":"Liz Touza, Julia","first_name":"Julia","last_name":"Liz Touza"},{"first_name":"Niklas","last_name":"Selfjord","full_name":"Selfjord, Niklas"},{"first_name":"Isabel","last_name":"Weisheit","full_name":"Weisheit, Isabel"},{"full_name":"Francescatto, Margherita","first_name":"Margherita","last_name":"Francescatto"},{"last_name":"Akcakaya","first_name":"Pinar","full_name":"Akcakaya, Pinar"},{"full_name":"Porritt, Michelle","last_name":"Porritt","first_name":"Michelle"},{"last_name":"Maresca","first_name":"Marcello","full_name":"Maresca, Marcello"},{"full_name":"Taylor, David","last_name":"Taylor","first_name":"David"},{"last_name":"Sienski","first_name":"Grzegorz","full_name":"Sienski, Grzegorz"}],"year":"2024"},{"author":[{"full_name":"Hibshman, Grace N.","first_name":"Grace N.","last_name":"Hibshman"},{"first_name":"Jack Peter Kelly","last_name":"Bravo","full_name":"Bravo, Jack Peter Kelly","orcid":"0000-0003-0456-0753","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e"},{"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."},{"full_name":"Johnson, Kenneth A.","last_name":"Johnson","first_name":"Kenneth A."},{"last_name":"Taylor","first_name":"David W.","full_name":"Taylor, David W."}],"year":"2024","date_updated":"2025-05-14T09:33:21Z","publisher":"Springer Nature","file":[{"file_name":"2024_NatureComm_Hibshman.pdf","creator":"dernst","file_id":"15386","relation":"main_file","access_level":"open_access","date_updated":"2024-05-13T11:46:19Z","success":1,"date_created":"2024-05-13T11:46:19Z","file_size":7477013,"checksum":"509c65919067a03ef8ad65c7192cd860","content_type":"application/pdf"}],"scopus_import":"1","has_accepted_license":"1","ddc":["570"],"citation":{"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.","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).","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>","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>.","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>"},"article_number":"3663","publication":"Nature Communications","status":"public","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","day":"30","volume":15,"oa":1,"article_type":"original","quality_controlled":"1","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"_id":"15372","date_published":"2024-04-30T00:00:00Z","language":[{"iso":"eng"}],"pmid":1,"intvolume":"        15","DOAJ_listed":"1","publication_status":"published","department":[{"_id":"JaBr"}],"oa_version":"Published Version","date_created":"2024-05-12T22:01:00Z","doi":"10.1038/s41467-024-47830-3","article_processing_charge":"Yes","title":"Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9","external_id":{"pmid":["38688943"]},"type":"journal_article","file_date_updated":"2024-05-13T11:46:19Z","month":"04","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."}],"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.","corr_author":"1","publication_identifier":{"eissn":["2041-1723"]}},{"article_processing_charge":"No","oa_version":"None","doi":"10.1016/j.bpj.2024.03.015","date_created":"2024-06-04T06:41:03Z","title":"Lipid droplets as substrates for protein phase separation","publication_identifier":{"issn":["0006-3495"]},"external_id":{"pmid":["38462838"]},"abstract":[{"text":"Membrane-associated protein phase separation plays critical roles in cell biology, driving essential cellular phenomena from immune signaling to membrane traffic. Importantly, by reducing dimensionality from three to two dimensions, lipid bilayers can nucleate phase separation at far lower concentrations compared with those required for phase separation in solution. How might other intracellular lipid substrates, such as lipid droplets, contribute to nucleation of phase separation? Distinct from bilayer membranes, lipid droplets consist of a phospholipid monolayer surrounding a core of neutral lipids, and they are energy storage organelles that protect cells from lipotoxicity and oxidative stress. Here, we show that intrinsically disordered proteins can undergo phase separation on the surface of synthetic and cell-derived lipid droplets. Specifically, we find that the model disordered domains FUS LC and LAF-1 RGG separate into protein-rich and protein-depleted phases on the surfaces of lipid droplets. Owing to the hydrophobic nature of interactions between FUS LC proteins, increasing ionic strength drives an increase in its phase separation on droplet surfaces. The opposite is true for LAF-1 RGG, owing to the electrostatic nature of its interprotein interactions. In both cases, protein-rich phases on the surfaces of synthetic and cell-derived lipid droplets demonstrate molecular mobility indicative of a liquid-like state. Our results show that lipid droplets can nucleate protein condensates, suggesting that protein phase separation could be key in organizing biological processes involving lipid droplets.","lang":"eng"}],"type":"journal_article","month":"06","date_published":"2024-06-04T00:00:00Z","_id":"17111","pmid":1,"publication_status":"published","intvolume":"       123","language":[{"iso":"eng"}],"issue":"11","volume":123,"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","day":"04","quality_controlled":"1","article_type":"original","extern":"1","date_updated":"2025-01-13T11:03:41Z","year":"2024","author":[{"first_name":"Advika","last_name":"Kamatar","full_name":"Kamatar, Advika"},{"id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753","first_name":"Jack Peter Kelly","last_name":"Bravo","full_name":"Bravo, Jack Peter Kelly"},{"first_name":"Feng","last_name":"Yuan","full_name":"Yuan, Feng"},{"last_name":"Wang","first_name":"Liping","full_name":"Wang, Liping"},{"full_name":"Lafer, Eileen M.","last_name":"Lafer","first_name":"Eileen M."},{"first_name":"David W.","last_name":"Taylor","full_name":"Taylor, David W."},{"first_name":"Jeanne C.","last_name":"Stachowiak","full_name":"Stachowiak, Jeanne C."},{"first_name":"Sapun H.","last_name":"Parekh","full_name":"Parekh, Sapun H."}],"page":"1494-1507","citation":{"apa":"Kamatar, A., Bravo, J. P. K., Yuan, F., Wang, L., Lafer, E. M., Taylor, D. W., … Parekh, S. H. (2024). Lipid droplets as substrates for protein phase separation. <i>Biophysical Journal</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.bpj.2024.03.015\">https://doi.org/10.1016/j.bpj.2024.03.015</a>","mla":"Kamatar, Advika, et al. “Lipid Droplets as Substrates for Protein Phase Separation.” <i>Biophysical Journal</i>, vol. 123, no. 11, Elsevier, 2024, pp. 1494–507, doi:<a href=\"https://doi.org/10.1016/j.bpj.2024.03.015\">10.1016/j.bpj.2024.03.015</a>.","ama":"Kamatar A, Bravo JPK, Yuan F, et al. Lipid droplets as substrates for protein phase separation. <i>Biophysical Journal</i>. 2024;123(11):1494-1507. doi:<a href=\"https://doi.org/10.1016/j.bpj.2024.03.015\">10.1016/j.bpj.2024.03.015</a>","ieee":"A. Kamatar <i>et al.</i>, “Lipid droplets as substrates for protein phase separation,” <i>Biophysical Journal</i>, vol. 123, no. 11. Elsevier, pp. 1494–1507, 2024.","ista":"Kamatar A, Bravo JPK, Yuan F, Wang L, Lafer EM, Taylor DW, Stachowiak JC, Parekh SH. 2024. Lipid droplets as substrates for protein phase separation. Biophysical Journal. 123(11), 1494–1507.","short":"A. Kamatar, J.P.K. Bravo, F. Yuan, L. Wang, E.M. Lafer, D.W. Taylor, J.C. Stachowiak, S.H. Parekh, Biophysical Journal 123 (2024) 1494–1507.","chicago":"Kamatar, Advika, Jack Peter Kelly Bravo, Feng Yuan, Liping Wang, Eileen M. Lafer, David W. Taylor, Jeanne C. Stachowiak, and Sapun H. Parekh. “Lipid Droplets as Substrates for Protein Phase Separation.” <i>Biophysical Journal</i>. Elsevier, 2024. <a href=\"https://doi.org/10.1016/j.bpj.2024.03.015\">https://doi.org/10.1016/j.bpj.2024.03.015</a>."},"publication":"Biophysical Journal","status":"public","scopus_import":"1","publisher":"Elsevier"},{"publication_status":"published","intvolume":"       383","pmid":1,"language":[{"iso":"eng"}],"date_published":"2024-02-01T00:00:00Z","_id":"17112","publication_identifier":{"eissn":["1095-9203"],"issn":["0036-8075"]},"acknowledgement":"We thank R. Fregoso Ocampo for assistance with negative-stain EM imaging. This work was funded by Dutch Research Council (NWO) VIDI grant VI.Vidi.203.074 (R.H.J.S.), NWO Spinoza grant SPI 93-537 (J.v.d.O.), European Research Council (ERC) Advanced grant ERC-AdG-834279 (J.v.d.O.), ERC CoG grant 817834 (T.J.G.E.), NWO VICI grant VI.C.192.016 (T.J.G.E.), Volkswagen Foundation grant 96725 (T.J.G.E.), National Institute of General Medical Sciences of the National Institutes of Health grant R35GM138348 (D.W.T.), Welch Foundation research grant F-1938 (D.W.T.), a Robert J. Kleberg, Jr. And Helen C. Kleberg Foundation medical research grant (D.W.T.), and American Cancer Society Research Scholar grant RSG-21-050-01-DMC (D.W.T.).","abstract":[{"lang":"eng","text":"The generation of cyclic oligoadenylates and subsequent allosteric activation of proteins that carry sensory domains is a distinctive feature of type III CRISPR-Cas systems. In this work, we characterize a set of associated genes of a type III-B system from Haliangium ochraceum that contains two caspase-like proteases, SAVED-CHAT and PCaspase (prokaryotic caspase), co-opted from a cyclic oligonucleotide–based antiphage signaling system (CBASS). Cyclic tri–adenosine monophosphate (AMP)–induced oligomerization of SAVED-CHAT activates proteolytic activity of the CHAT domains, which specifically cleave and activate PCaspase. Subsequently, activated PCaspase cleaves a multitude of proteins, which results in a strong interference phenotype in vivo in Escherichia coli. Taken together, our findings reveal how a CRISPR-Cas–based detection of a target RNA triggers a cascade of caspase-associated proteolytic activities."}],"month":"02","type":"journal_article","external_id":{"pmid":["38301007"]},"title":"Type III-B CRISPR-Cas cascade of proteolytic cleavages","article_processing_charge":"No","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1101/2023.06.23.546230"}],"doi":"10.1126/science.adk0378","date_created":"2024-06-04T06:41:26Z","oa_version":"Preprint","publication":"Science","status":"public","page":"512-519","citation":{"short":"J.A. Steens, J.P.K. Bravo, C.R.P. Salazar, C. Yildiz, A.M. Amieiro, S. Köstlbacher, S.H.P. Prinsen, C. Patinios, A. Bardis, A. Barendregt, R.A. Scheltema, T.J.G. Ettema, J. van der Oost, D.W. Taylor, R.H.J. Staals, Science 383 (2024) 512–519.","ista":"Steens JA, Bravo JPK, Salazar CRP, Yildiz C, Amieiro AM, Köstlbacher S, Prinsen SHP, Patinios C, Bardis A, Barendregt A, Scheltema RA, Ettema TJG, van der Oost J, Taylor DW, Staals RHJ. 2024. Type III-B CRISPR-Cas cascade of proteolytic cleavages. Science. 383(6682), 512–519.","chicago":"Steens, Jurre A., Jack Peter Kelly Bravo, Carl Raymund P. Salazar, Caglar Yildiz, Afonso M. Amieiro, Stephan Köstlbacher, Stijn H.P. Prinsen, et al. “Type III-B CRISPR-Cas Cascade of Proteolytic Cleavages.” <i>Science</i>. American Association for the Advancement of Science, 2024. <a href=\"https://doi.org/10.1126/science.adk0378\">https://doi.org/10.1126/science.adk0378</a>.","ama":"Steens JA, Bravo JPK, Salazar CRP, et al. Type III-B CRISPR-Cas cascade of proteolytic cleavages. <i>Science</i>. 2024;383(6682):512-519. doi:<a href=\"https://doi.org/10.1126/science.adk0378\">10.1126/science.adk0378</a>","mla":"Steens, Jurre A., et al. “Type III-B CRISPR-Cas Cascade of Proteolytic Cleavages.” <i>Science</i>, vol. 383, no. 6682, American Association for the Advancement of Science, 2024, pp. 512–19, doi:<a href=\"https://doi.org/10.1126/science.adk0378\">10.1126/science.adk0378</a>.","ieee":"J. A. Steens <i>et al.</i>, “Type III-B CRISPR-Cas cascade of proteolytic cleavages,” <i>Science</i>, vol. 383, no. 6682. American Association for the Advancement of Science, pp. 512–519, 2024.","apa":"Steens, J. A., Bravo, J. P. K., Salazar, C. R. P., Yildiz, C., Amieiro, A. M., Köstlbacher, S., … Staals, R. H. J. (2024). Type III-B CRISPR-Cas cascade of proteolytic cleavages. <i>Science</i>. American Association for the Advancement of Science. <a href=\"https://doi.org/10.1126/science.adk0378\">https://doi.org/10.1126/science.adk0378</a>"},"OA_type":"green","scopus_import":"1","publisher":"American Association for the Advancement of Science","date_updated":"2025-09-24T08:31:45Z","extern":"1","year":"2024","author":[{"last_name":"Steens","first_name":"Jurre A.","full_name":"Steens, Jurre A."},{"id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753","full_name":"Bravo, Jack Peter Kelly","last_name":"Bravo","first_name":"Jack Peter Kelly"},{"last_name":"Salazar","first_name":"Carl Raymund P.","full_name":"Salazar, Carl Raymund P."},{"first_name":"Caglar","last_name":"Yildiz","full_name":"Yildiz, Caglar"},{"last_name":"Amieiro","first_name":"Afonso M.","full_name":"Amieiro, Afonso M."},{"last_name":"Köstlbacher","first_name":"Stephan","full_name":"Köstlbacher, Stephan"},{"full_name":"Prinsen, Stijn H.P.","first_name":"Stijn H.P.","last_name":"Prinsen"},{"last_name":"Patinios","first_name":"Constantinos","full_name":"Patinios, Constantinos"},{"full_name":"Bardis, Andreas","last_name":"Bardis","first_name":"Andreas"},{"first_name":"Arjan","last_name":"Barendregt","full_name":"Barendregt, Arjan"},{"full_name":"Scheltema, Richard A.","first_name":"Richard A.","last_name":"Scheltema"},{"last_name":"Ettema","first_name":"Thijs J.G.","full_name":"Ettema, Thijs J.G."},{"full_name":"van der Oost, John","last_name":"van der Oost","first_name":"John"},{"last_name":"Taylor","first_name":"David W.","full_name":"Taylor, David W."},{"first_name":"Raymond H.J.","last_name":"Staals","full_name":"Staals, Raymond H.J."}],"quality_controlled":"1","article_type":"original","oa":1,"volume":383,"issue":"6682","day":"01","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","OA_place":"repository"},{"intvolume":"        15","publication_status":"published","pmid":1,"language":[{"iso":"eng"}],"date_published":"2024-04-30T00:00:00Z","_id":"17113","publication_identifier":{"issn":["2041-1723"]},"month":"04","type":"journal_article","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 <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."}],"external_id":{"pmid":["38688943"]},"title":"Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9","oa_version":"Published Version","date_created":"2024-06-04T06:42:07Z","doi":"10.1038/s41467-024-47830-3","article_processing_charge":"Yes","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1038/s41467-024-47830-3"}],"publication":"Nature Communications","status":"public","article_number":"3663","citation":{"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.","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>","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).","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."},"publisher":"Springer Nature","scopus_import":"1","date_updated":"2024-10-14T12:34:26Z","extern":"1","author":[{"full_name":"Hibshman, Grace N.","last_name":"Hibshman","first_name":"Grace N."},{"last_name":"Bravo","first_name":"Jack Peter Kelly","full_name":"Bravo, Jack Peter Kelly","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753"},{"full_name":"Hooper, Matthew M.","last_name":"Hooper","first_name":"Matthew M."},{"first_name":"Tyler L.","last_name":"Dangerfield","full_name":"Dangerfield, Tyler L."},{"full_name":"Zhang, Hongshan","last_name":"Zhang","first_name":"Hongshan"},{"full_name":"Finkelstein, Ilya J.","first_name":"Ilya J.","last_name":"Finkelstein"},{"first_name":"Kenneth A.","last_name":"Johnson","full_name":"Johnson, Kenneth A."},{"last_name":"Taylor","first_name":"David W.","full_name":"Taylor, David W."}],"year":"2024","article_type":"original","quality_controlled":"1","oa":1,"volume":15,"day":"30","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87"},{"publisher":"Springer Nature","scopus_import":"1","status":"public","publication":"Nature Communications","citation":{"ieee":"E. A. Schwartz <i>et al.</i>, “RNA targeting and cleavage by the type III-Dv CRISPR effector complex,” <i>Nature Communications</i>, vol. 15. Springer Nature, 2024.","ama":"Schwartz EA, Bravo JPK, Ahsan M, et al. RNA targeting and cleavage by the type III-Dv CRISPR effector complex. <i>Nature Communications</i>. 2024;15. doi:<a href=\"https://doi.org/10.1038/s41467-024-47506-y\">10.1038/s41467-024-47506-y</a>","apa":"Schwartz, E. A., Bravo, J. P. K., Ahsan, M., Macias, L. A., McCafferty, C. L., Dangerfield, T. L., … Taylor, D. W. (2024). RNA targeting and cleavage by the type III-Dv CRISPR effector complex. <i>Nature Communications</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41467-024-47506-y\">https://doi.org/10.1038/s41467-024-47506-y</a>","mla":"Schwartz, Evan A., et al. “RNA Targeting and Cleavage by the Type III-Dv CRISPR Effector Complex.” <i>Nature Communications</i>, vol. 15, 3324, Springer Nature, 2024, doi:<a href=\"https://doi.org/10.1038/s41467-024-47506-y\">10.1038/s41467-024-47506-y</a>.","chicago":"Schwartz, Evan A., Jack Peter Kelly Bravo, Mohd Ahsan, Luis A. Macias, Caitlyn L. McCafferty, Tyler L. Dangerfield, Jada N. Walker, et al. “RNA Targeting and Cleavage by the Type III-Dv CRISPR Effector Complex.” <i>Nature Communications</i>. Springer Nature, 2024. <a href=\"https://doi.org/10.1038/s41467-024-47506-y\">https://doi.org/10.1038/s41467-024-47506-y</a>.","ista":"Schwartz EA, Bravo JPK, Ahsan M, Macias LA, McCafferty CL, Dangerfield TL, Walker JN, Brodbelt JS, Palermo G, Fineran PC, Fagerlund RD, Taylor DW. 2024. RNA targeting and cleavage by the type III-Dv CRISPR effector complex. Nature Communications. 15, 3324.","short":"E.A. Schwartz, J.P.K. Bravo, M. Ahsan, L.A. Macias, C.L. McCafferty, T.L. Dangerfield, J.N. Walker, J.S. Brodbelt, G. Palermo, P.C. Fineran, R.D. Fagerlund, D.W. Taylor, Nature Communications 15 (2024)."},"article_number":"3324","author":[{"full_name":"Schwartz, Evan A.","first_name":"Evan A.","last_name":"Schwartz"},{"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"},{"full_name":"Ahsan, Mohd","last_name":"Ahsan","first_name":"Mohd"},{"full_name":"Macias, Luis A.","first_name":"Luis A.","last_name":"Macias"},{"last_name":"McCafferty","first_name":"Caitlyn L.","full_name":"McCafferty, Caitlyn L."},{"full_name":"Dangerfield, Tyler L.","last_name":"Dangerfield","first_name":"Tyler L."},{"first_name":"Jada N.","last_name":"Walker","full_name":"Walker, Jada N."},{"first_name":"Jennifer S.","last_name":"Brodbelt","full_name":"Brodbelt, Jennifer S."},{"full_name":"Palermo, Giulia","last_name":"Palermo","first_name":"Giulia"},{"full_name":"Fineran, Peter C.","last_name":"Fineran","first_name":"Peter C."},{"full_name":"Fagerlund, Robert D.","first_name":"Robert D.","last_name":"Fagerlund"},{"first_name":"David W.","last_name":"Taylor","full_name":"Taylor, David W."}],"year":"2024","date_updated":"2024-06-04T07:05:26Z","extern":"1","article_type":"original","quality_controlled":"1","oa":1,"day":"18","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","volume":15,"language":[{"iso":"eng"}],"intvolume":"        15","publication_status":"published","pmid":1,"_id":"17114","date_published":"2024-04-18T00:00:00Z","month":"04","type":"journal_article","abstract":[{"text":"CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction<jats:sup>1–5</jats:sup>. Target RNA cleavage at regular intervals is characteristic of type III effector complexes<jats:sup>6–8</jats:sup>. Here, we determine the structures of the <jats:italic>Synechocystis</jats:italic> type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors<jats:sup>9,10</jats:sup>, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2′-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.","lang":"eng"}],"external_id":{"pmid":["38637512"]},"publication_identifier":{"issn":["2041-1723"]},"title":"RNA targeting and cleavage by the type III-Dv CRISPR effector complex","oa_version":"Published Version","doi":"10.1038/s41467-024-47506-y","date_created":"2024-06-04T06:43:02Z","main_file_link":[{"url":"https://doi.org/10.1038/s41467-024-47506-y","open_access":"1"}],"article_processing_charge":"Yes"},{"language":[{"iso":"eng"}],"pmid":1,"publication_status":"published","intvolume":"       630","_id":"17442","date_published":"2024-06-27T00:00:00Z","external_id":{"pmid":["38740055"]},"abstract":[{"lang":"eng","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"}],"month":"06","type":"journal_article","corr_author":"1","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.).","publication_identifier":{"issn":["0028-0836"],"eissn":["1476-4687"]},"department":[{"_id":"JaBr"}],"article_processing_charge":"No","main_file_link":[{"url":"https://pmc.ncbi.nlm.nih.gov/articles/PMC11649018/","open_access":"1"}],"doi":"10.1038/s41586-024-07515-9","oa_version":"Submitted Version","date_created":"2024-08-19T09:41:18Z","title":"Plasmid targeting and destruction by the DdmDE bacterial defence system","scopus_import":"1","publisher":"Springer Nature","page":"961-967","OA_type":"green","citation":{"short":"J.P.K. Bravo, D.A. Ramos, R. Fregoso Ocampo, C. Ingram, D.W. Taylor, Nature 630 (2024) 961–967.","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.","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>.","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>","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.","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>"},"publication":"Nature","status":"public","year":"2024","author":[{"full_name":"Bravo, Jack Peter Kelly","first_name":"Jack Peter Kelly","last_name":"Bravo","orcid":"0000-0003-0456-0753","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e"},{"full_name":"Ramos, Delisa A.","first_name":"Delisa A.","last_name":"Ramos"},{"full_name":"Fregoso Ocampo, Rodrigo","last_name":"Fregoso Ocampo","first_name":"Rodrigo"},{"first_name":"Caiden","last_name":"Ingram","full_name":"Ingram, Caiden"},{"first_name":"David W.","last_name":"Taylor","full_name":"Taylor, David W."}],"date_updated":"2025-06-24T12:47:21Z","oa":1,"quality_controlled":"1","article_type":"original","OA_place":"repository","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","day":"27","issue":"8018","volume":630},{"date_published":"2024-10-01T00:00:00Z","isi":1,"_id":"17494","publication_status":"published","intvolume":"        19","pmid":1,"language":[{"iso":"eng"}],"title":"Anti-plasmid immunity: A key to pathogen success?","main_file_link":[{"url":"https://doi.org/10.1080/17460913.2024.2389720","open_access":"1"}],"article_processing_charge":"No","doi":"10.1080/17460913.2024.2389720","date_created":"2024-09-05T07:32:00Z","oa_version":"Published Version","department":[{"_id":"JaBr"}],"publication_identifier":{"issn":["1746-0913"],"eissn":["1746-0921"]},"corr_author":"1","acknowledgement":"I would like to thank K Kiernan for insightful comments and feedback. J P K Bravo is supported by IST Austria.","month":"10","type":"journal_article","external_id":{"pmid":["39230568"],"isi":["001306115400001"]},"date_updated":"2025-09-08T09:03:00Z","year":"2024","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"}],"publication":"Future Microbiology","status":"public","OA_type":"free access","page":"1269-1272","citation":{"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>.","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>","short":"J.P.K. Bravo, Future Microbiology 19 (2024) 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>.","ista":"Bravo JPK. 2024. Anti-plasmid immunity: A key to pathogen success? Future Microbiology. 19(15), 1269–1272."},"has_accepted_license":"1","scopus_import":"1","publisher":"Taylor & Francis","volume":19,"issue":"15","day":"01","OA_place":"publisher","user_id":"317138e5-6ab7-11ef-aa6d-ffef3953e345","quality_controlled":"1","article_type":"letter_note","oa":1},{"article_type":"original","quality_controlled":"1","oa":1,"volume":83,"issue":"5","day":"02","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","status":"public","publication":"Molecular Cell","page":"746-758.e5","citation":{"chicago":"O’Brien, Roisin E., Jack Peter Kelly Bravo, Delisa Ramos, Grace N. Hibshman, Jacquelyn T. Wright, and David W. Taylor. “Structural Snapshots of R-Loop Formation by a Type I-C CRISPR Cascade.” <i>Molecular Cell</i>. Elsevier, 2023. <a href=\"https://doi.org/10.1016/j.molcel.2023.01.024\">https://doi.org/10.1016/j.molcel.2023.01.024</a>.","short":"R.E. O’Brien, J.P.K. Bravo, D. Ramos, G.N. Hibshman, J.T. Wright, D.W. Taylor, Molecular Cell 83 (2023) 746–758.e5.","ista":"O’Brien RE, Bravo JPK, Ramos D, Hibshman GN, Wright JT, Taylor DW. 2023. Structural snapshots of R-loop formation by a type I-C CRISPR Cascade. Molecular Cell. 83(5), 746–758.e5.","apa":"O’Brien, R. E., Bravo, J. P. K., Ramos, D., Hibshman, G. N., Wright, J. T., &#38; Taylor, D. W. (2023). Structural snapshots of R-loop formation by a type I-C CRISPR Cascade. <i>Molecular Cell</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.molcel.2023.01.024\">https://doi.org/10.1016/j.molcel.2023.01.024</a>","ama":"O’Brien RE, Bravo JPK, Ramos D, Hibshman GN, Wright JT, Taylor DW. Structural snapshots of R-loop formation by a type I-C CRISPR Cascade. <i>Molecular Cell</i>. 2023;83(5):746-758.e5. doi:<a href=\"https://doi.org/10.1016/j.molcel.2023.01.024\">10.1016/j.molcel.2023.01.024</a>","ieee":"R. E. O’Brien, J. P. K. Bravo, D. Ramos, G. N. Hibshman, J. T. Wright, and D. W. Taylor, “Structural snapshots of R-loop formation by a type I-C CRISPR Cascade,” <i>Molecular Cell</i>, vol. 83, no. 5. Elsevier, p. 746–758.e5, 2023.","mla":"O’Brien, Roisin E., et al. “Structural Snapshots of R-Loop Formation by a Type I-C CRISPR Cascade.” <i>Molecular Cell</i>, vol. 83, no. 5, Elsevier, 2023, p. 746–758.e5, doi:<a href=\"https://doi.org/10.1016/j.molcel.2023.01.024\">10.1016/j.molcel.2023.01.024</a>."},"publisher":"Elsevier","scopus_import":"1","date_updated":"2024-06-04T06:33:54Z","extern":"1","author":[{"full_name":"O’Brien, Roisin E.","last_name":"O’Brien","first_name":"Roisin E."},{"id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753","full_name":"Bravo, Jack Peter Kelly","last_name":"Bravo","first_name":"Jack Peter Kelly"},{"full_name":"Ramos, Delisa","last_name":"Ramos","first_name":"Delisa"},{"first_name":"Grace N.","last_name":"Hibshman","full_name":"Hibshman, Grace N."},{"last_name":"Wright","first_name":"Jacquelyn T.","full_name":"Wright, Jacquelyn T."},{"full_name":"Taylor, David W.","last_name":"Taylor","first_name":"David W."}],"year":"2023","publication_identifier":{"issn":["1097-2765"]},"type":"journal_article","month":"03","abstract":[{"lang":"eng","text":"Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. Here, we present structures of D. vulgaris type I-C Cascade at various stages of double-stranded (ds)DNA target capture, revealing mechanisms that underpin PAM recognition and Cascade allosteric activation. We uncover an interesting mechanism of non-target strand (NTS) DNA stabilization via stacking interactions with the “belly” subunits, securing the NTS in place. This “molecular seatbelt” mechanism facilitates efficient R-loop formation and prevents dsDNA reannealing. Additionally, we provide structural insights into how two anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking PAM scanning. These observations form a structural basis for directional R-loop formation and reveal how different Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity."}],"external_id":{"pmid":["36805026"]},"title":"Structural snapshots of R-loop formation by a type I-C CRISPR Cascade","doi":"10.1016/j.molcel.2023.01.024","oa_version":"Published Version","date_created":"2024-03-20T10:40:56Z","article_processing_charge":"Yes (in subscription journal)","main_file_link":[{"url":"https://doi.org/10.1016/j.molcel.2023.01.024","open_access":"1"}],"intvolume":"        83","publication_status":"published","pmid":1,"language":[{"iso":"eng"}],"date_published":"2023-03-02T00:00:00Z","_id":"15129","keyword":["Cell Biology","Molecular Biology"]},{"intvolume":"       613","publication_status":"published","pmid":1,"language":[{"iso":"eng"}],"date_published":"2023-01-04T00:00:00Z","_id":"15130","publication_identifier":{"issn":["0028-0836"],"eissn":["1476-4687"]},"month":"01","type":"journal_article","abstract":[{"text":"Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection<jats:sup>1</jats:sup>. Here we report structures of Cas12a2 in binary, ternary and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of ‘aromatic clamp’ residues that are crucial for double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.","lang":"eng"}],"external_id":{"pmid":["36599980"]},"title":"RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2","oa_version":"Published Version","doi":"10.1038/s41586-022-05560-w","date_created":"2024-03-20T10:41:36Z","article_processing_charge":"Yes (in subscription journal)","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1038/s41586-022-05560-w"}],"publication":"Nature","status":"public","citation":{"chicago":"Bravo, Jack Peter Kelly, Thomson Hallmark, Bronson Naegle, Chase L. Beisel, Ryan N. Jackson, and David W. Taylor. “RNA Targeting Unleashes Indiscriminate Nuclease Activity of CRISPR–Cas12a2.” <i>Nature</i>. Springer Nature, 2023. <a href=\"https://doi.org/10.1038/s41586-022-05560-w\">https://doi.org/10.1038/s41586-022-05560-w</a>.","short":"J.P.K. Bravo, T. Hallmark, B. Naegle, C.L. Beisel, R.N. Jackson, D.W. Taylor, Nature 613 (2023) 582–587.","ista":"Bravo JPK, Hallmark T, Naegle B, Beisel CL, Jackson RN, Taylor DW. 2023. RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2. Nature. 613(7944), 582–587.","ieee":"J. P. K. Bravo, T. Hallmark, B. Naegle, C. L. Beisel, R. N. Jackson, and D. W. Taylor, “RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2,” <i>Nature</i>, vol. 613, no. 7944. Springer Nature, pp. 582–587, 2023.","mla":"Bravo, Jack Peter Kelly, et al. “RNA Targeting Unleashes Indiscriminate Nuclease Activity of CRISPR–Cas12a2.” <i>Nature</i>, vol. 613, no. 7944, Springer Nature, 2023, pp. 582–87, doi:<a href=\"https://doi.org/10.1038/s41586-022-05560-w\">10.1038/s41586-022-05560-w</a>.","apa":"Bravo, J. P. K., Hallmark, T., Naegle, B., Beisel, C. L., Jackson, R. N., &#38; Taylor, D. W. (2023). RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2. <i>Nature</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41586-022-05560-w\">https://doi.org/10.1038/s41586-022-05560-w</a>","ama":"Bravo JPK, Hallmark T, Naegle B, Beisel CL, Jackson RN, Taylor DW. RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2. <i>Nature</i>. 2023;613(7944):582-587. doi:<a href=\"https://doi.org/10.1038/s41586-022-05560-w\">10.1038/s41586-022-05560-w</a>"},"page":"582-587","publisher":"Springer Nature","scopus_import":"1","date_updated":"2024-06-04T06:30:59Z","extern":"1","author":[{"first_name":"Jack Peter Kelly","last_name":"Bravo","full_name":"Bravo, Jack Peter Kelly","orcid":"0000-0003-0456-0753","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e"},{"first_name":"Thomson","last_name":"Hallmark","full_name":"Hallmark, Thomson"},{"last_name":"Naegle","first_name":"Bronson","full_name":"Naegle, Bronson"},{"full_name":"Beisel, Chase L.","first_name":"Chase L.","last_name":"Beisel"},{"full_name":"Jackson, Ryan N.","first_name":"Ryan N.","last_name":"Jackson"},{"last_name":"Taylor","first_name":"David W.","full_name":"Taylor, David W."}],"year":"2023","article_type":"original","quality_controlled":"1","oa":1,"volume":613,"issue":"7944","day":"04","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87"},{"title":"A single 2′-O-methylation of ribosomal RNA gates assembly of a functional ribosome","main_file_link":[{"url":"https://doi.org/10.1038/s41594-022-00891-8","open_access":"1"}],"article_processing_charge":"Yes (in subscription journal)","oa_version":"Published Version","date_created":"2024-03-20T10:41:45Z","doi":"10.1038/s41594-022-00891-8","abstract":[{"text":"RNA modifications are widespread in biology and abundant in ribosomal RNA. However, the importance of these modifications is not well understood. We show that methylation of a single nucleotide, in the catalytic center of the large subunit, gates ribosome assembly. Massively parallel mutational scanning of the essential nuclear GTPase Nog2 identified important interactions with rRNA, particularly with the 2′-<jats:italic>O</jats:italic>-methylated A-site base Gm2922. We found that methylation of G2922 is needed for assembly and efficient nuclear export of the large subunit. Critically, we identified single amino acid changes in Nog2 that completely bypass dependence on G2922 methylation and used cryoelectron microscopy to directly visualize how methylation flips Gm2922 into the active site channel of Nog2. This work demonstrates that a single RNA modification is a critical checkpoint in ribosome biogenesis, suggesting that such modifications can play an important role in regulation and assembly of macromolecular machines.","lang":"eng"}],"type":"journal_article","month":"12","external_id":{"pmid":["36536102"]},"publication_identifier":{"eissn":["1545-9985"],"issn":["1545-9993"]},"_id":"15131","keyword":["Molecular Biology","Structural Biology"],"date_published":"2022-12-19T00:00:00Z","language":[{"iso":"eng"}],"publication_status":"published","intvolume":"        30","pmid":1,"day":"19","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","volume":30,"quality_controlled":"1","article_type":"original","oa":1,"year":"2022","author":[{"first_name":"James N.","last_name":"Yelland","full_name":"Yelland, James N."},{"first_name":"Jack Peter Kelly","last_name":"Bravo","full_name":"Bravo, Jack Peter Kelly","orcid":"0000-0003-0456-0753","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e"},{"last_name":"Black","first_name":"Joshua J.","full_name":"Black, Joshua J."},{"first_name":"David W.","last_name":"Taylor","full_name":"Taylor, David W."},{"first_name":"Arlen W.","last_name":"Johnson","full_name":"Johnson, Arlen W."}],"date_updated":"2024-06-04T06:27:09Z","extern":"1","scopus_import":"1","publisher":"Springer Nature","status":"public","publication":"Nature Structural & Molecular Biology","page":"91-98","citation":{"apa":"Yelland, J. N., Bravo, J. P. K., Black, J. J., Taylor, D. W., &#38; Johnson, A. W. (2022). A single 2′-O-methylation of ribosomal RNA gates assembly of a functional ribosome. <i>Nature Structural &#38; Molecular Biology</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41594-022-00891-8\">https://doi.org/10.1038/s41594-022-00891-8</a>","mla":"Yelland, James N., et al. “A Single 2′-O-Methylation of Ribosomal RNA Gates Assembly of a Functional Ribosome.” <i>Nature Structural &#38; Molecular Biology</i>, vol. 30, Springer Nature, 2022, pp. 91–98, doi:<a href=\"https://doi.org/10.1038/s41594-022-00891-8\">10.1038/s41594-022-00891-8</a>.","ama":"Yelland JN, Bravo JPK, Black JJ, Taylor DW, Johnson AW. A single 2′-O-methylation of ribosomal RNA gates assembly of a functional ribosome. <i>Nature Structural &#38; Molecular Biology</i>. 2022;30:91-98. doi:<a href=\"https://doi.org/10.1038/s41594-022-00891-8\">10.1038/s41594-022-00891-8</a>","ieee":"J. N. Yelland, J. P. K. Bravo, J. J. Black, D. W. Taylor, and A. W. Johnson, “A single 2′-O-methylation of ribosomal RNA gates assembly of a functional ribosome,” <i>Nature Structural &#38; Molecular Biology</i>, vol. 30. Springer Nature, pp. 91–98, 2022.","ista":"Yelland JN, Bravo JPK, Black JJ, Taylor DW, Johnson AW. 2022. A single 2′-O-methylation of ribosomal RNA gates assembly of a functional ribosome. Nature Structural &#38; Molecular Biology. 30, 91–98.","chicago":"Yelland, James N., Jack Peter Kelly Bravo, Joshua J. Black, David W. Taylor, and Arlen W. Johnson. “A Single 2′-O-Methylation of Ribosomal RNA Gates Assembly of a Functional Ribosome.” <i>Nature Structural &#38; Molecular Biology</i>. Springer Nature, 2022. <a href=\"https://doi.org/10.1038/s41594-022-00891-8\">https://doi.org/10.1038/s41594-022-00891-8</a>.","short":"J.N. Yelland, J.P.K. Bravo, J.J. Black, D.W. Taylor, A.W. Johnson, Nature Structural &#38; Molecular Biology 30 (2022) 91–98."}},{"language":[{"iso":"eng"}],"intvolume":"        78","publication_status":"published","pmid":1,"_id":"15132","keyword":["Biomedical Engineering","Bioengineering","Biotechnology"],"date_published":"2022-12-01T00:00:00Z","month":"12","type":"journal_article","abstract":[{"lang":"eng","text":"Clustered regularly interspaced short palindromic repeats - CRISPR-associated protein (CRISPR-Cas) systems are a critical component of the bacterial adaptive immune response. Since the discovery that they can be reengineered as programmable RNA-guided nucleases, there has been significant interest in using these systems to perform diverse and precise genetic manipulations. Here, we outline recent advances in the mechanistic understanding of CRISPR-Cas9, how these findings have been leveraged in the rational redesign of Cas9 variants with altered activities, and how these novel tools can be exploited for biotechnology and therapeutics. We also discuss the potential of the ubiquitous, yet often-overlooked, multisubunit CRISPR effector complexes for large-scale genomic deletions. Furthermore, we highlight how future structural studies will bolster these technologies."}],"external_id":{"pmid":["36371895"]},"publication_identifier":{"issn":["0958-1669"]},"title":"Constructing next-generation CRISPR–Cas tools from structural blueprints","oa_version":"None","doi":"10.1016/j.copbio.2022.102839","date_created":"2024-03-20T10:41:53Z","article_processing_charge":"No","publisher":"Elsevier","scopus_import":"1","status":"public","publication":"Current Opinion in Biotechnology","citation":{"apa":"Bravo, J. P. K., Hibshman, G. N., &#38; Taylor, D. W. (2022). Constructing next-generation CRISPR–Cas tools from structural blueprints. <i>Current Opinion in Biotechnology</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.copbio.2022.102839\">https://doi.org/10.1016/j.copbio.2022.102839</a>","ieee":"J. P. K. Bravo, G. N. Hibshman, and D. W. Taylor, “Constructing next-generation CRISPR–Cas tools from structural blueprints,” <i>Current Opinion in Biotechnology</i>, vol. 78. Elsevier, 2022.","ama":"Bravo JPK, Hibshman GN, Taylor DW. Constructing next-generation CRISPR–Cas tools from structural blueprints. <i>Current Opinion in Biotechnology</i>. 2022;78. doi:<a href=\"https://doi.org/10.1016/j.copbio.2022.102839\">10.1016/j.copbio.2022.102839</a>","mla":"Bravo, Jack Peter Kelly, et al. “Constructing Next-Generation CRISPR–Cas Tools from Structural Blueprints.” <i>Current Opinion in Biotechnology</i>, vol. 78, 102839, Elsevier, 2022, doi:<a href=\"https://doi.org/10.1016/j.copbio.2022.102839\">10.1016/j.copbio.2022.102839</a>.","short":"J.P.K. Bravo, G.N. Hibshman, D.W. Taylor, Current Opinion in Biotechnology 78 (2022).","ista":"Bravo JPK, Hibshman GN, Taylor DW. 2022. Constructing next-generation CRISPR–Cas tools from structural blueprints. Current Opinion in Biotechnology. 78, 102839.","chicago":"Bravo, Jack Peter Kelly, Grace N Hibshman, and David W Taylor. “Constructing Next-Generation CRISPR–Cas Tools from Structural Blueprints.” <i>Current Opinion in Biotechnology</i>. Elsevier, 2022. <a href=\"https://doi.org/10.1016/j.copbio.2022.102839\">https://doi.org/10.1016/j.copbio.2022.102839</a>."},"article_number":"102839","author":[{"last_name":"Bravo","first_name":"Jack Peter Kelly","full_name":"Bravo, Jack Peter Kelly","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753"},{"first_name":"Grace N","last_name":"Hibshman","full_name":"Hibshman, Grace N"},{"full_name":"Taylor, David W","last_name":"Taylor","first_name":"David W"}],"year":"2022","date_updated":"2024-10-14T12:34:11Z","extern":"1","article_type":"review","quality_controlled":"1","day":"01","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","volume":78},{"publication_identifier":{"issn":["2041-1723"]},"abstract":[{"lang":"eng","text":"In the evolutionary arms race against phage, bacteria have assembled a diverse arsenal of antiviral immune strategies. While the recently discovered DISARM (Defense Island System Associated with Restriction-Modification) systems can provide protection against a wide range of phage, the molecular mechanisms that underpin broad antiviral targeting but avoiding autoimmunity remain enigmatic. Here, we report cryo-EM structures of the core DISARM complex, DrmAB, both alone and in complex with an unmethylated phage DNA mimetic. These structures reveal that DrmAB core complex is autoinhibited by a trigger loop (TL) within DrmA and binding to DNA substrates containing a 5′ overhang dislodges the TL, initiating a long-range structural rearrangement for DrmAB activation. Together with structure-guided in vivo studies, our work provides insights into the mechanism of phage DNA recognition and specific activation of this widespread antiviral defense system."}],"month":"05","type":"journal_article","external_id":{"pmid":["35624106"]},"title":"Structural basis for broad anti-phage immunity by DISARM","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1038/s41467-022-30673-1"}],"article_processing_charge":"Yes","date_created":"2024-03-20T10:41:59Z","oa_version":"Published Version","doi":"10.1038/s41467-022-30673-1","publication_status":"published","intvolume":"        13","pmid":1,"language":[{"iso":"eng"}],"date_published":"2022-05-27T00:00:00Z","_id":"15133","keyword":["General Physics and Astronomy","General Biochemistry","Genetics and Molecular Biology","General Chemistry","Multidisciplinary"],"quality_controlled":"1","article_type":"original","oa":1,"volume":13,"day":"27","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","status":"public","publication":"Nature Communications","article_number":"2987","citation":{"short":"J.P.K. Bravo, C. Aparicio-Maldonado, F.L. Nobrega, S.J.J. Brouns, D.W. Taylor, Nature Communications 13 (2022).","chicago":"Bravo, Jack Peter Kelly, Cristian Aparicio-Maldonado, Franklin L. Nobrega, Stan J. J. Brouns, and David W. Taylor. “Structural Basis for Broad Anti-Phage Immunity by DISARM.” <i>Nature Communications</i>. Springer Nature, 2022. <a href=\"https://doi.org/10.1038/s41467-022-30673-1\">https://doi.org/10.1038/s41467-022-30673-1</a>.","ista":"Bravo JPK, Aparicio-Maldonado C, Nobrega FL, Brouns SJJ, Taylor DW. 2022. Structural basis for broad anti-phage immunity by DISARM. Nature Communications. 13, 2987.","mla":"Bravo, Jack Peter Kelly, et al. “Structural Basis for Broad Anti-Phage Immunity by DISARM.” <i>Nature Communications</i>, vol. 13, 2987, Springer Nature, 2022, doi:<a href=\"https://doi.org/10.1038/s41467-022-30673-1\">10.1038/s41467-022-30673-1</a>.","apa":"Bravo, J. P. K., Aparicio-Maldonado, C., Nobrega, F. L., Brouns, S. J. J., &#38; Taylor, D. W. (2022). Structural basis for broad anti-phage immunity by DISARM. <i>Nature Communications</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41467-022-30673-1\">https://doi.org/10.1038/s41467-022-30673-1</a>","ama":"Bravo JPK, Aparicio-Maldonado C, Nobrega FL, Brouns SJJ, Taylor DW. Structural basis for broad anti-phage immunity by DISARM. <i>Nature Communications</i>. 2022;13. doi:<a href=\"https://doi.org/10.1038/s41467-022-30673-1\">10.1038/s41467-022-30673-1</a>","ieee":"J. P. K. Bravo, C. Aparicio-Maldonado, F. L. Nobrega, S. J. J. Brouns, and D. W. Taylor, “Structural basis for broad anti-phage immunity by DISARM,” <i>Nature Communications</i>, vol. 13. Springer Nature, 2022."},"scopus_import":"1","publisher":"Springer Nature","date_updated":"2024-06-04T06:16:38Z","extern":"1","year":"2022","author":[{"full_name":"Bravo, Jack Peter Kelly","first_name":"Jack Peter Kelly","last_name":"Bravo","orcid":"0000-0003-0456-0753","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e"},{"full_name":"Aparicio-Maldonado, Cristian","first_name":"Cristian","last_name":"Aparicio-Maldonado"},{"last_name":"Nobrega","first_name":"Franklin L.","full_name":"Nobrega, Franklin L."},{"full_name":"Brouns, Stan J. J.","first_name":"Stan J. J.","last_name":"Brouns"},{"last_name":"Taylor","first_name":"David W.","full_name":"Taylor, David W."}]},{"article_number":"2829","citation":{"chicago":"Schwartz, Evan A., Tess M. McBride, Jack Peter Kelly Bravo, Daniel Wrapp, Peter C. Fineran, Robert D. Fagerlund, and David W. Taylor. “Structural Rearrangements Allow Nucleic Acid Discrimination by Type I-D Cascade.” <i>Nature Communications</i>. Springer Nature, 2022. <a href=\"https://doi.org/10.1038/s41467-022-30402-8\">https://doi.org/10.1038/s41467-022-30402-8</a>.","ista":"Schwartz EA, McBride TM, Bravo JPK, Wrapp D, Fineran PC, Fagerlund RD, Taylor DW. 2022. Structural rearrangements allow nucleic acid discrimination by type I-D Cascade. Nature Communications. 13, 2829.","short":"E.A. Schwartz, T.M. McBride, J.P.K. Bravo, D. Wrapp, P.C. Fineran, R.D. Fagerlund, D.W. Taylor, Nature Communications 13 (2022).","ama":"Schwartz EA, McBride TM, Bravo JPK, et al. Structural rearrangements allow nucleic acid discrimination by type I-D Cascade. <i>Nature Communications</i>. 2022;13. doi:<a href=\"https://doi.org/10.1038/s41467-022-30402-8\">10.1038/s41467-022-30402-8</a>","mla":"Schwartz, Evan A., et al. “Structural Rearrangements Allow Nucleic Acid Discrimination by Type I-D Cascade.” <i>Nature Communications</i>, vol. 13, 2829, Springer Nature, 2022, doi:<a href=\"https://doi.org/10.1038/s41467-022-30402-8\">10.1038/s41467-022-30402-8</a>.","apa":"Schwartz, E. A., McBride, T. M., Bravo, J. P. K., Wrapp, D., Fineran, P. C., Fagerlund, R. D., &#38; Taylor, D. W. (2022). Structural rearrangements allow nucleic acid discrimination by type I-D Cascade. <i>Nature Communications</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41467-022-30402-8\">https://doi.org/10.1038/s41467-022-30402-8</a>","ieee":"E. A. Schwartz <i>et al.</i>, “Structural rearrangements allow nucleic acid discrimination by type I-D Cascade,” <i>Nature Communications</i>, vol. 13. Springer Nature, 2022."},"publication":"Nature Communications","status":"public","scopus_import":"1","publisher":"Springer Nature","extern":"1","date_updated":"2024-06-04T06:14:28Z","year":"2022","author":[{"last_name":"Schwartz","first_name":"Evan A.","full_name":"Schwartz, Evan A."},{"full_name":"McBride, Tess M.","last_name":"McBride","first_name":"Tess M."},{"id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","orcid":"0000-0003-0456-0753","full_name":"Bravo, Jack Peter Kelly","last_name":"Bravo","first_name":"Jack Peter Kelly"},{"full_name":"Wrapp, Daniel","last_name":"Wrapp","first_name":"Daniel"},{"full_name":"Fineran, Peter C.","last_name":"Fineran","first_name":"Peter C."},{"last_name":"Fagerlund","first_name":"Robert D.","full_name":"Fagerlund, Robert D."},{"first_name":"David W.","last_name":"Taylor","full_name":"Taylor, David W."}],"oa":1,"quality_controlled":"1","article_type":"original","volume":13,"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","day":"20","pmid":1,"publication_status":"published","intvolume":"        13","language":[{"iso":"eng"}],"date_published":"2022-05-20T00:00:00Z","keyword":["General Physics and Astronomy","General Biochemistry","Genetics and Molecular Biology","General Chemistry","Multidisciplinary"],"_id":"15134","publication_identifier":{"issn":["2041-1723"]},"external_id":{"pmid":["35595728"]},"abstract":[{"text":"CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3′ binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.","lang":"eng"}],"month":"05","type":"journal_article","article_processing_charge":"Yes","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1038/s41467-022-30402-8"}],"date_created":"2024-03-20T10:42:05Z","oa_version":"Published Version","doi":"10.1038/s41467-022-30402-8","title":"Structural rearrangements allow nucleic acid discrimination by type I-D Cascade"},{"abstract":[{"lang":"eng","text":"CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1,2,3,4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5,6,7. Although Cas9 variants with greater discrimination against mismatches have been designed8,9,10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants."}],"type":"journal_article","month":"03","external_id":{"pmid":["35236982"]},"publication_identifier":{"eissn":["1476-4687"],"issn":["0028-0836"]},"title":"Structural basis for mismatch surveillance by CRISPR–Cas9","main_file_link":[{"url":"https://doi.org/10.1038/s41586-022-04470-1","open_access":"1"}],"article_processing_charge":"Yes (in subscription journal)","doi":"10.1038/s41586-022-04470-1","oa_version":"Published Version","date_created":"2024-03-20T10:42:21Z","language":[{"iso":"eng"}],"publication_status":"published","intvolume":"       603","pmid":1,"related_material":{"link":[{"relation":"erratum","url":"https://doi.org/10.1038/s41586-022-04655-8"}]},"_id":"15136","date_published":"2022-03-02T00:00:00Z","quality_controlled":"1","article_type":"original","oa":1,"day":"02","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","volume":603,"issue":"7900","scopus_import":"1","publisher":"Springer Nature","status":"public","publication":"Nature","citation":{"ama":"Bravo JPK, Liu M-S, Hibshman GN, et al. Structural basis for mismatch surveillance by CRISPR–Cas9. <i>Nature</i>. 2022;603(7900):343-347. doi:<a href=\"https://doi.org/10.1038/s41586-022-04470-1\">10.1038/s41586-022-04470-1</a>","mla":"Bravo, Jack Peter Kelly, et al. “Structural Basis for Mismatch Surveillance by CRISPR–Cas9.” <i>Nature</i>, vol. 603, no. 7900, Springer Nature, 2022, pp. 343–47, doi:<a href=\"https://doi.org/10.1038/s41586-022-04470-1\">10.1038/s41586-022-04470-1</a>.","ieee":"J. P. K. Bravo <i>et al.</i>, “Structural basis for mismatch surveillance by CRISPR–Cas9,” <i>Nature</i>, vol. 603, no. 7900. Springer Nature, pp. 343–347, 2022.","apa":"Bravo, J. P. K., Liu, M.-S., Hibshman, G. N., Dangerfield, T. L., Jung, K., McCool, R. S., … Taylor, D. W. (2022). Structural basis for mismatch surveillance by CRISPR–Cas9. <i>Nature</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41586-022-04470-1\">https://doi.org/10.1038/s41586-022-04470-1</a>","ista":"Bravo JPK, Liu M-S, Hibshman GN, Dangerfield TL, Jung K, McCool RS, Johnson KA, Taylor DW. 2022. Structural basis for mismatch surveillance by CRISPR–Cas9. Nature. 603(7900), 343–347.","chicago":"Bravo, Jack Peter Kelly, Mu-Sen Liu, Grace N. Hibshman, Tyler L. Dangerfield, Kyungseok Jung, Ryan S. McCool, Kenneth A. Johnson, and David W. Taylor. “Structural Basis for Mismatch Surveillance by CRISPR–Cas9.” <i>Nature</i>. Springer Nature, 2022. <a href=\"https://doi.org/10.1038/s41586-022-04470-1\">https://doi.org/10.1038/s41586-022-04470-1</a>.","short":"J.P.K. Bravo, M.-S. Liu, G.N. Hibshman, T.L. Dangerfield, K. Jung, R.S. McCool, K.A. Johnson, D.W. Taylor, Nature 603 (2022) 343–347."},"page":"343-347","year":"2022","author":[{"first_name":"Jack Peter Kelly","last_name":"Bravo","full_name":"Bravo, Jack Peter Kelly","orcid":"0000-0003-0456-0753","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e"},{"first_name":"Mu-Sen","last_name":"Liu","full_name":"Liu, Mu-Sen"},{"first_name":"Grace N.","last_name":"Hibshman","full_name":"Hibshman, Grace N."},{"first_name":"Tyler L.","last_name":"Dangerfield","full_name":"Dangerfield, Tyler L."},{"full_name":"Jung, Kyungseok","first_name":"Kyungseok","last_name":"Jung"},{"first_name":"Ryan S.","last_name":"McCool","full_name":"McCool, Ryan S."},{"full_name":"Johnson, Kenneth A.","last_name":"Johnson","first_name":"Kenneth A."},{"full_name":"Taylor, David W.","first_name":"David W.","last_name":"Taylor"}],"date_updated":"2024-06-04T06:36:59Z","extern":"1"},{"publication_identifier":{"issn":["1935-472X"],"eissn":["1937-8661"]},"quality_controlled":"1","type":"journal_article","month":"04","article_type":"letter_note","volume":42,"title":"SuperFi-Cas9 exceeds fidelity, matches speed of original Cas9","article_processing_charge":"No","doi":"10.1089/gen.42.04.03","date_created":"2024-03-20T10:43:19Z","oa_version":"None","issue":"4","day":"01","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","publication_status":"published","publication":"Genetic Engineering & Biotechnology News","status":"public","intvolume":"        42","citation":{"ista":"Bravo JPK. 2022. SuperFi-Cas9 exceeds fidelity, matches speed of original Cas9. Genetic Engineering &#38; Biotechnology News. 42(4), 12.","chicago":"Bravo, Jack Peter Kelly. “SuperFi-Cas9 Exceeds Fidelity, Matches Speed of Original Cas9.” <i>Genetic Engineering &#38; Biotechnology News</i>. Mary Ann Liebert, 2022. <a href=\"https://doi.org/10.1089/gen.42.04.03\">https://doi.org/10.1089/gen.42.04.03</a>.","short":"J.P.K. Bravo, Genetic Engineering &#38; Biotechnology News 42 (2022) 12.","mla":"Bravo, Jack Peter Kelly. “SuperFi-Cas9 Exceeds Fidelity, Matches Speed of Original Cas9.” <i>Genetic Engineering &#38; Biotechnology News</i>, vol. 42, no. 4, Mary Ann Liebert, 2022, p. 12, doi:<a href=\"https://doi.org/10.1089/gen.42.04.03\">10.1089/gen.42.04.03</a>.","ieee":"J. P. K. Bravo, “SuperFi-Cas9 exceeds fidelity, matches speed of original Cas9,” <i>Genetic Engineering &#38; Biotechnology News</i>, vol. 42, no. 4. Mary Ann Liebert, p. 12, 2022.","ama":"Bravo JPK. SuperFi-Cas9 exceeds fidelity, matches speed of original Cas9. <i>Genetic Engineering &#38; Biotechnology News</i>. 2022;42(4):12. doi:<a href=\"https://doi.org/10.1089/gen.42.04.03\">10.1089/gen.42.04.03</a>","apa":"Bravo, J. P. K. (2022). SuperFi-Cas9 exceeds fidelity, matches speed of original Cas9. <i>Genetic Engineering &#38; Biotechnology News</i>. Mary Ann Liebert. <a href=\"https://doi.org/10.1089/gen.42.04.03\">https://doi.org/10.1089/gen.42.04.03</a>"},"page":"12","scopus_import":"1","publisher":"Mary Ann Liebert","language":[{"iso":"eng"}],"date_updated":"2024-10-14T12:32:14Z","date_published":"2022-04-01T00:00:00Z","extern":"1","_id":"15144","year":"2022","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"}],"keyword":["Management of Technology and Innovation","Biomedical Engineering","Bioengineering","Biotechnology"]},{"_id":"17115","year":"2022","author":[{"first_name":"Roisin E.","last_name":"O’Brien","full_name":"O’Brien, Roisin E."},{"orcid":"0000-0003-0456-0753","id":"96aecfa5-8931-11ee-af30-aa6a5d6eee0e","last_name":"Bravo","first_name":"Jack Peter Kelly","full_name":"Bravo, Jack Peter Kelly"},{"full_name":"Ramos, Delisa","first_name":"Delisa","last_name":"Ramos"},{"full_name":"Hibshman, Grace N.","first_name":"Grace N.","last_name":"Hibshman"},{"full_name":"Wright, Jacquelyn T.","first_name":"Jacquelyn T.","last_name":"Wright"},{"last_name":"Taylor","first_name":"David W.","full_name":"Taylor, David W."}],"date_updated":"2024-06-04T07:03:02Z","date_published":"2022-06-15T00:00:00Z","extern":"1","publisher":"Cold Spring Harbor Laboratory","language":[{"iso":"eng"}],"publication_status":"published","publication":"bioRxiv","status":"public","citation":{"chicago":"O’Brien, Roisin E., Jack Peter Kelly Bravo, Delisa Ramos, Grace N. Hibshman, Jacquelyn T. Wright, and David W. Taylor. “Modes of Inhibition Used by Phage Anti-CRISPRs to Evade Type I-C Cascade.” <i>BioRxiv</i>. Cold Spring Harbor Laboratory, 2022. <a href=\"https://doi.org/10.1101/2022.06.15.496202\">https://doi.org/10.1101/2022.06.15.496202</a>.","ista":"O’Brien RE, Bravo JPK, Ramos D, Hibshman GN, Wright JT, Taylor DW. 2022. Modes of inhibition used by phage anti-CRISPRs to evade type I-C Cascade. bioRxiv, <a href=\"https://doi.org/10.1101/2022.06.15.496202\">10.1101/2022.06.15.496202</a>.","short":"R.E. O’Brien, J.P.K. Bravo, D. Ramos, G.N. Hibshman, J.T. Wright, D.W. Taylor, BioRxiv (2022).","ieee":"R. E. O’Brien, J. P. K. Bravo, D. Ramos, G. N. Hibshman, J. T. Wright, and D. W. Taylor, “Modes of inhibition used by phage anti-CRISPRs to evade type I-C Cascade,” <i>bioRxiv</i>. Cold Spring Harbor Laboratory, 2022.","mla":"O’Brien, Roisin E., et al. “Modes of Inhibition Used by Phage Anti-CRISPRs to Evade Type I-C Cascade.” <i>BioRxiv</i>, Cold Spring Harbor Laboratory, 2022, doi:<a href=\"https://doi.org/10.1101/2022.06.15.496202\">10.1101/2022.06.15.496202</a>.","apa":"O’Brien, R. E., Bravo, J. P. K., Ramos, D., Hibshman, G. N., Wright, J. T., &#38; Taylor, D. W. (2022). Modes of inhibition used by phage anti-CRISPRs to evade type I-C Cascade. <i>bioRxiv</i>. Cold Spring Harbor Laboratory. <a href=\"https://doi.org/10.1101/2022.06.15.496202\">https://doi.org/10.1101/2022.06.15.496202</a>","ama":"O’Brien RE, Bravo JPK, Ramos D, Hibshman GN, Wright JT, Taylor DW. Modes of inhibition used by phage anti-CRISPRs to evade type I-C Cascade. <i>bioRxiv</i>. 2022. doi:<a href=\"https://doi.org/10.1101/2022.06.15.496202\">10.1101/2022.06.15.496202</a>"},"day":"15","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","title":"Modes of inhibition used by phage anti-CRISPRs to evade type I-C Cascade","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1101/2022.06.15.496202"}],"article_processing_charge":"No","oa_version":"Preprint","date_created":"2024-06-04T06:43:30Z","doi":"10.1101/2022.06.15.496202","abstract":[{"text":"Cascades are RNA-guided multi-subunit CRISPR-Cas surveillances complexes that target foreign nucleic acids for destruction. Here, we present a 2.9-Å resolution cryo-electron (cryo-EM) structure of the <jats:italic>D. vulgaris</jats:italic> type I-C Cascade bound to a double-stranded (ds)DNA target. Our data shows how the 5’-TTC-3’ protospacer adjacent motif (PAM) sequence is recognized, and provides a unique mechanism through which the displaced, single-stranded non-target strand (NTS) is stabilized via stacking interactions with protein subunits in order to favor R-loop formation and prevent dsDNA re-annealing. Additionally, we provide structural insights into how diverse anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking initial DNA binding. These observations provide a structural basis for directional R-loop formation and reveal how divergent Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.","lang":"eng"}],"month":"06","type":"preprint","oa":1}]