@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{18545,
  abstract     = {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.},
  author       = {Degtev, Dmitrii and Bravo, Jack Peter Kelly and Emmanouilidi, Aikaterini and Zdravković, Aleksandar and Choong, Oi Kuan and Liz Touza, Julia and Selfjord, Niklas and Weisheit, Isabel and Francescatto, Margherita and Akcakaya, Pinar and Porritt, Michelle and Maresca, Marcello and Taylor, David and Sienski, Grzegorz},
  issn         = {2041-1723},
  journal      = {Nature Communications},
  publisher    = {Springer Nature},
  title        = {{Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles}},
  doi          = {10.1038/s41467-024-53418-8},
  volume       = {15},
  year         = {2024},
}

@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{17111,
  abstract     = {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.},
  author       = {Kamatar, Advika and Bravo, Jack Peter Kelly and Yuan, Feng and Wang, Liping and Lafer, Eileen M. and Taylor, David W. and Stachowiak, Jeanne C. and Parekh, Sapun H.},
  issn         = {0006-3495},
  journal      = {Biophysical Journal},
  number       = {11},
  pages        = {1494--1507},
  publisher    = {Elsevier},
  title        = {{Lipid droplets as substrates for protein phase separation}},
  doi          = {10.1016/j.bpj.2024.03.015},
  volume       = {123},
  year         = {2024},
}

@article{17112,
  abstract     = {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.},
  author       = {Steens, Jurre A. and Bravo, Jack Peter Kelly and Salazar, Carl Raymund P. and Yildiz, Caglar and Amieiro, Afonso M. and Köstlbacher, Stephan and Prinsen, Stijn H.P. and Patinios, Constantinos and Bardis, Andreas and Barendregt, Arjan and Scheltema, Richard A. and Ettema, Thijs J.G. and van der Oost, John and Taylor, David W. and Staals, Raymond H.J.},
  issn         = {1095-9203},
  journal      = {Science},
  number       = {6682},
  pages        = {512--519},
  publisher    = {American Association for the Advancement of Science},
  title        = {{Type III-B CRISPR-Cas cascade of proteolytic cleavages}},
  doi          = {10.1126/science.adk0378},
  volume       = {383},
  year         = {2024},
}

@article{17113,
  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 <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.},
  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{17114,
  abstract     = {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.},
  author       = {Schwartz, Evan A. and Bravo, Jack Peter Kelly and Ahsan, Mohd and Macias, Luis A. and McCafferty, Caitlyn L. and Dangerfield, Tyler L. and Walker, Jada N. and Brodbelt, Jennifer S. and Palermo, Giulia and Fineran, Peter C. and Fagerlund, Robert D. and Taylor, David W.},
  issn         = {2041-1723},
  journal      = {Nature Communications},
  publisher    = {Springer Nature},
  title        = {{RNA targeting and cleavage by the type III-Dv CRISPR effector complex}},
  doi          = {10.1038/s41467-024-47506-y},
  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},
}

@article{15129,
  abstract     = {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.},
  author       = {O’Brien, Roisin E. and Bravo, Jack Peter Kelly and Ramos, Delisa and Hibshman, Grace N. and Wright, Jacquelyn T. and Taylor, David W.},
  issn         = {1097-2765},
  journal      = {Molecular Cell},
  keywords     = {Cell Biology, Molecular Biology},
  number       = {5},
  pages        = {746--758.e5},
  publisher    = {Elsevier},
  title        = {{Structural snapshots of R-loop formation by a type I-C CRISPR Cascade}},
  doi          = {10.1016/j.molcel.2023.01.024},
  volume       = {83},
  year         = {2023},
}

@article{15130,
  abstract     = {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.},
  author       = {Bravo, Jack Peter Kelly and Hallmark, Thomson and Naegle, Bronson and Beisel, Chase L. and Jackson, Ryan N. and Taylor, David W.},
  issn         = {1476-4687},
  journal      = {Nature},
  number       = {7944},
  pages        = {582--587},
  publisher    = {Springer Nature},
  title        = {{RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2}},
  doi          = {10.1038/s41586-022-05560-w},
  volume       = {613},
  year         = {2023},
}

@article{15131,
  abstract     = {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.},
  author       = {Yelland, James N. and Bravo, Jack Peter Kelly and Black, Joshua J. and Taylor, David W. and Johnson, Arlen W.},
  issn         = {1545-9985},
  journal      = {Nature Structural & Molecular Biology},
  keywords     = {Molecular Biology, Structural Biology},
  pages        = {91--98},
  publisher    = {Springer Nature},
  title        = {{A single 2′-O-methylation of ribosomal RNA gates assembly of a functional ribosome}},
  doi          = {10.1038/s41594-022-00891-8},
  volume       = {30},
  year         = {2022},
}

@article{15132,
  abstract     = {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.},
  author       = {Bravo, Jack Peter Kelly and Hibshman, Grace N and Taylor, David W},
  issn         = {0958-1669},
  journal      = {Current Opinion in Biotechnology},
  keywords     = {Biomedical Engineering, Bioengineering, Biotechnology},
  publisher    = {Elsevier},
  title        = {{Constructing next-generation CRISPR–Cas tools from structural blueprints}},
  doi          = {10.1016/j.copbio.2022.102839},
  volume       = {78},
  year         = {2022},
}

@article{15133,
  abstract     = {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.},
  author       = {Bravo, Jack Peter Kelly and Aparicio-Maldonado, Cristian and Nobrega, Franklin L. and Brouns, Stan J. J. and Taylor, David W.},
  issn         = {2041-1723},
  journal      = {Nature Communications},
  keywords     = {General Physics and Astronomy, General Biochemistry, Genetics and Molecular Biology, General Chemistry, Multidisciplinary},
  publisher    = {Springer Nature},
  title        = {{Structural basis for broad anti-phage immunity by DISARM}},
  doi          = {10.1038/s41467-022-30673-1},
  volume       = {13},
  year         = {2022},
}

@article{15134,
  abstract     = {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.},
  author       = {Schwartz, Evan A. and McBride, Tess M. and Bravo, Jack Peter Kelly and Wrapp, Daniel and Fineran, Peter C. and Fagerlund, Robert D. and Taylor, David W.},
  issn         = {2041-1723},
  journal      = {Nature Communications},
  keywords     = {General Physics and Astronomy, General Biochemistry, Genetics and Molecular Biology, General Chemistry, Multidisciplinary},
  publisher    = {Springer Nature},
  title        = {{Structural rearrangements allow nucleic acid discrimination by type I-D Cascade}},
  doi          = {10.1038/s41467-022-30402-8},
  volume       = {13},
  year         = {2022},
}

@article{15136,
  abstract     = {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.},
  author       = {Bravo, Jack Peter Kelly and Liu, Mu-Sen and Hibshman, Grace N. and Dangerfield, Tyler L. and Jung, Kyungseok and McCool, Ryan S. and Johnson, Kenneth A. and Taylor, David W.},
  issn         = {1476-4687},
  journal      = {Nature},
  number       = {7900},
  pages        = {343--347},
  publisher    = {Springer Nature},
  title        = {{Structural basis for mismatch surveillance by CRISPR–Cas9}},
  doi          = {10.1038/s41586-022-04470-1},
  volume       = {603},
  year         = {2022},
}

@article{15144,
  author       = {Bravo, Jack Peter Kelly},
  issn         = {1937-8661},
  journal      = {Genetic Engineering & Biotechnology News},
  keywords     = {Management of Technology and Innovation, Biomedical Engineering, Bioengineering, Biotechnology},
  number       = {4},
  pages        = {12},
  publisher    = {Mary Ann Liebert},
  title        = {{SuperFi-Cas9 exceeds fidelity, matches speed of original Cas9}},
  doi          = {10.1089/gen.42.04.03},
  volume       = {42},
  year         = {2022},
}

@unpublished{17115,
  abstract     = {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.},
  author       = {O’Brien, Roisin E. and Bravo, Jack Peter Kelly and Ramos, Delisa and Hibshman, Grace N. and Wright, Jacquelyn T. and Taylor, David W.},
  booktitle    = {bioRxiv},
  publisher    = {Cold Spring Harbor Laboratory},
  title        = {{Modes of inhibition used by phage anti-CRISPRs to evade type I-C Cascade}},
  doi          = {10.1101/2022.06.15.496202},
  year         = {2022},
}