@article{20963,
  abstract     = {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.},
  author       = {Dmytrenko, Oleg and Yuan, Biao and Crosby, Kadin T. and Krebel, Max and Chen, Xiye and Nowak, Jakub S. and Chramiec-Głąbik, Andrzej and Filani, Bamidele and Gribling-Burrer, Anne-Sophie and van der Toorn, Wiep and von Kleist, Max and Achmedov, Tatjana and Smyth, Redmond P. and Glatt, Sebastian and Bravo, Jack Peter Kelly and Heinz, Dirk W. and Jackson, Ryan N. and Beisel, Chase L.},
  issn         = {1476-4687},
  journal      = {Nature},
  publisher    = {Springer Nature},
  title        = {{RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity}},
  doi          = {10.1038/s41586-025-09852-9},
  year         = {2026},
}

@article{20143,
  abstract     = {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.},
  author       = {Zhang, Zhiying and Todeschini, Thomas C. and Wu, Yi and Kogay, Roman and Naji, Ameena and Cardenas Rodriguez, Joaquin and Mondi, Rupavidhya and Kaganovich, Daniel and Taylor, David W. and Bravo, Jack Peter Kelly and Teplova, Marianna and Amen, Triana and Koonin, Eugene and Patel, Dinshaw J. and Nobrega, Franklin L.},
  issn         = {1097-4172},
  journal      = {Cell},
  number       = {21},
  pages        = {5862--5877.e23},
  publisher    = {Elsevier},
  title        = {{Kiwa is a membrane-embedded defense supercomplex activated at phage attachment sites}},
  doi          = {10.1016/j.cell.2025.07.002},
  volume       = {188},
  year         = {2025},
}

@article{18848,
  abstract     = {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.},
  author       = {Ocampo, Rodrigo Fregoso and Bravo, Jack Peter Kelly and Dangerfield, Tyler L. and Nocedal, Isabel and Jirde, Samatar A. and Alexander, Lisa M. and Thomas, Nicole C. and Das, Anjali and Nielson, Sarah and Johnson, Kenneth A. and Brown, Christopher T. and Butterfield, Cristina N. and Goltsman, Daniela S.A. and Taylor, David W.},
  issn         = {2041-1723},
  journal      = {Nature Communications},
  publisher    = {Springer Nature},
  title        = {{DNA targeting by compact Cas9d and its resurrected ancestor}},
  doi          = {10.1038/s41467-024-55573-4},
  volume       = {16},
  year         = {2025},
}

@article{15372,
  abstract     = {CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.},
  author       = {Hibshman, Grace N. and Bravo, Jack Peter Kelly and Hooper, Matthew M. and Dangerfield, Tyler L. and Zhang, Hongshan and Finkelstein, Ilya J. and Johnson, Kenneth A. and Taylor, David W.},
  issn         = {2041-1723},
  journal      = {Nature Communications},
  publisher    = {Springer Nature},
  title        = {{Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9}},
  doi          = {10.1038/s41467-024-47830-3},
  volume       = {15},
  year         = {2024},
}

@article{17442,
  abstract     = {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.

},
  author       = {Bravo, Jack Peter Kelly and Ramos, Delisa A. and Fregoso Ocampo, Rodrigo and Ingram, Caiden and Taylor, David W.},
  issn         = {1476-4687},
  journal      = {Nature},
  number       = {8018},
  pages        = {961--967},
  publisher    = {Springer Nature},
  title        = {{Plasmid targeting and destruction by the DdmDE bacterial defence system}},
  doi          = {10.1038/s41586-024-07515-9},
  volume       = {630},
  year         = {2024},
}

@article{17494,
  author       = {Bravo, Jack Peter Kelly},
  issn         = {1746-0921},
  journal      = {Future Microbiology},
  number       = {15},
  pages        = {1269--1272},
  publisher    = {Taylor & Francis},
  title        = {{Anti-plasmid immunity: A key to pathogen success?}},
  doi          = {10.1080/17460913.2024.2389720},
  volume       = {19},
  year         = {2024},
}