---
OA_place: publisher
OA_type: hybrid
PlanS_conform: '1'
_id: '20963'
abstract:
- lang: eng
  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.
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).'
article_processing_charge: Yes (via OA deal)
article_type: original
author:
- first_name: Oleg
  full_name: Dmytrenko, Oleg
  last_name: Dmytrenko
- first_name: Biao
  full_name: Yuan, Biao
  last_name: Yuan
- first_name: Kadin T.
  full_name: Crosby, Kadin T.
  last_name: Crosby
- first_name: Max
  full_name: Krebel, Max
  last_name: Krebel
- first_name: Xiye
  full_name: Chen, Xiye
  last_name: Chen
- first_name: Jakub S.
  full_name: Nowak, Jakub S.
  last_name: Nowak
- first_name: Andrzej
  full_name: Chramiec-Głąbik, Andrzej
  last_name: Chramiec-Głąbik
- first_name: Bamidele
  full_name: Filani, Bamidele
  last_name: Filani
- first_name: Anne-Sophie
  full_name: Gribling-Burrer, Anne-Sophie
  last_name: Gribling-Burrer
- first_name: Wiep
  full_name: van der Toorn, Wiep
  last_name: van der Toorn
- first_name: Max
  full_name: von Kleist, Max
  last_name: von Kleist
- first_name: Tatjana
  full_name: Achmedov, Tatjana
  last_name: Achmedov
- first_name: Redmond P.
  full_name: Smyth, Redmond P.
  last_name: Smyth
- first_name: Sebastian
  full_name: Glatt, Sebastian
  last_name: Glatt
- first_name: Jack Peter Kelly
  full_name: Bravo, Jack Peter Kelly
  id: 96aecfa5-8931-11ee-af30-aa6a5d6eee0e
  last_name: Bravo
  orcid: 0000-0003-0456-0753
- first_name: Dirk W.
  full_name: Heinz, Dirk W.
  last_name: Heinz
- first_name: Ryan N.
  full_name: Jackson, Ryan N.
  last_name: Jackson
- first_name: Chase L.
  full_name: Beisel, Chase L.
  last_name: Beisel
citation:
  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>
  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>
  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>.
  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.
  mla: Dmytrenko, Oleg, et al. “RNA-Triggered Cas12a3 Cleaves TRNA Tails to Execute
    Bacterial Immunity.” <i>Nature</i>, Springer Nature, 2026, doi:<a href="https://doi.org/10.1038/s41586-025-09852-9">10.1038/s41586-025-09852-9</a>.
  short: O. Dmytrenko, B. Yuan, K.T. Crosby, M. Krebel, X. Chen, J.S. Nowak, A. Chramiec-Głąbik,
    B. Filani, A.-S. Gribling-Burrer, W. van der Toorn, M. von Kleist, T. Achmedov,
    R.P. Smyth, S. Glatt, J.P.K. Bravo, D.W. Heinz, R.N. Jackson, C.L. Beisel, Nature
    (2026).
date_created: 2026-01-08T07:57:17Z
date_published: 2026-01-07T00:00:00Z
date_updated: 2026-01-12T10:13:56Z
day: '07'
ddc:
- '570'
department:
- _id: JaBr
doi: 10.1038/s41586-025-09852-9
external_id:
  pmid:
  - '41501459'
has_accepted_license: '1'
language:
- iso: eng
main_file_link:
- open_access: '1'
  url: https://doi.org/10.1038/s41586-025-09852-9
month: '01'
oa: 1
oa_version: Published Version
pmid: 1
publication: Nature
publication_identifier:
  eissn:
  - 1476-4687
  issn:
  - 0028-0836
publication_status: epub_ahead
publisher: Springer Nature
quality_controlled: '1'
scopus_import: '1'
status: public
title: RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity
tmp:
  image: /images/cc_by.png
  legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
  name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
  short: CC BY (4.0)
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
year: '2026'
...
---
OA_place: publisher
OA_type: hybrid
PlanS_conform: '1'
_id: '20143'
abstract:
- lang: eng
  text: Bacteria and archaea deploy diverse antiviral defense systems, many of which
    remain mechanistically uncharacterized. Here, we characterize Kiwa, a widespread
    two-component system composed of the transmembrane sensor KwaA and the DNA-binding
    effector KwaB. Cryogenic electron microscopy (cryo-EM) analysis reveals that KwaA
    and KwaB assemble into a large, membrane-associated supercomplex. Upon phage binding,
    KwaA senses infection at the membrane, leading to KwaB binding of ejected phage
    DNA and inhibition of replication and late transcription, without inducing host
    cell death. Although KwaB can bind DNA independently, its antiviral activity requires
    association with KwaA, suggesting spatial or conformational regulation. We show
    that the phage-encoded DNA-mimic protein Gam directly binds and inhibits KwaB
    but that co-expression with the Gam-targeted RecBCD system restores protection
    by Kiwa. Our findings support a model in which Kiwa coordinates membrane-associated
    detection of phage infection with downstream DNA binding by its effector, forming
    a spatially coordinated antiviral mechanism.
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).
article_processing_charge: Yes (in subscription journal)
article_type: original
author:
- first_name: Zhiying
  full_name: Zhang, Zhiying
  last_name: Zhang
- first_name: Thomas C.
  full_name: Todeschini, Thomas C.
  last_name: Todeschini
- first_name: Yi
  full_name: Wu, Yi
  last_name: Wu
- first_name: Roman
  full_name: Kogay, Roman
  last_name: Kogay
- first_name: Ameena
  full_name: Naji, Ameena
  last_name: Naji
- first_name: Joaquin
  full_name: Cardenas Rodriguez, Joaquin
  last_name: Cardenas Rodriguez
- first_name: Rupavidhya
  full_name: Mondi, Rupavidhya
  last_name: Mondi
- first_name: Daniel
  full_name: Kaganovich, Daniel
  last_name: Kaganovich
- first_name: David W.
  full_name: Taylor, David W.
  last_name: Taylor
- first_name: Jack Peter Kelly
  full_name: Bravo, Jack Peter Kelly
  id: 96aecfa5-8931-11ee-af30-aa6a5d6eee0e
  last_name: Bravo
  orcid: 0000-0003-0456-0753
- first_name: Marianna
  full_name: Teplova, Marianna
  last_name: Teplova
- first_name: Triana
  full_name: Amen, Triana
  last_name: Amen
- first_name: Eugene
  full_name: Koonin, Eugene
  last_name: Koonin
- first_name: Dinshaw J.
  full_name: Patel, Dinshaw J.
  last_name: Patel
- first_name: Franklin L.
  full_name: Nobrega, Franklin L.
  last_name: Nobrega
citation:
  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>
  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>
  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>.
  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.
  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.
  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>.
  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.
date_created: 2025-08-07T05:00:04Z
date_published: 2025-10-16T00:00:00Z
date_updated: 2025-12-29T14:15:58Z
day: '16'
ddc:
- '570'
department:
- _id: JaBr
doi: 10.1016/j.cell.2025.07.002
external_id:
  isi:
  - '001603560700005'
  pmid:
  - '40730155'
file:
- access_level: open_access
  checksum: b944de5fbd7455f58e1ff338ad352239
  content_type: application/pdf
  creator: dernst
  date_created: 2025-12-29T14:15:25Z
  date_updated: 2025-12-29T14:15:25Z
  file_id: '20875'
  file_name: 2025_Cell_Zhang.pdf
  file_size: 32104588
  relation: main_file
  success: 1
file_date_updated: 2025-12-29T14:15:25Z
has_accepted_license: '1'
intvolume: '       188'
isi: 1
issue: '21'
language:
- iso: eng
month: '10'
oa: 1
oa_version: Published Version
page: 5862-5877.e23
pmid: 1
publication: Cell
publication_identifier:
  eissn:
  - 1097-4172
  issn:
  - 0092-8674
publication_status: published
publisher: Elsevier
quality_controlled: '1'
scopus_import: '1'
status: public
title: Kiwa is a membrane-embedded defense supercomplex activated at phage attachment
  sites
tmp:
  image: /images/cc_by.png
  legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
  name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
  short: CC BY (4.0)
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 188
year: '2025'
...
---
DOAJ_listed: '1'
OA_place: publisher
OA_type: gold
_id: '18848'
abstract:
- lang: eng
  text: Type II CRISPR endonucleases are widely used programmable genome editing tools.
    Recently, CRISPR-Cas systems with highly compact nucleases have been discovered,
    including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures
    of a Cas9d nuclease (747 amino acids in length) in multiple functional states,
    revealing a stepwise process of DNA targeting involving a conformational switch
    in a REC2 domain insertion. Our structures provide insights into the intricately
    folded guide RNA which acts as a structural scaffold to anchor small, flexible
    protein domains for DNA recognition. The sgRNA can be truncated by up to ~25%
    yet still retain activity in vivo. Using ancestral sequence reconstruction, we
    generated compact nucleases capable of efficient genome editing in mammalian cells.
    Collectively, our results provide mechanistic insights into the evolution and
    DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for
    future re-engineering of minimal RNA-guided DNA endonucleases.
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.).
article_number: '457'
article_processing_charge: Yes
article_type: original
author:
- first_name: Rodrigo Fregoso
  full_name: Ocampo, Rodrigo Fregoso
  last_name: Ocampo
- first_name: Jack Peter Kelly
  full_name: Bravo, Jack Peter Kelly
  id: 96aecfa5-8931-11ee-af30-aa6a5d6eee0e
  last_name: Bravo
  orcid: 0000-0003-0456-0753
- first_name: Tyler L.
  full_name: Dangerfield, Tyler L.
  last_name: Dangerfield
- first_name: Isabel
  full_name: Nocedal, Isabel
  last_name: Nocedal
- first_name: Samatar A.
  full_name: Jirde, Samatar A.
  last_name: Jirde
- first_name: Lisa M.
  full_name: Alexander, Lisa M.
  last_name: Alexander
- first_name: Nicole C.
  full_name: Thomas, Nicole C.
  last_name: Thomas
- first_name: Anjali
  full_name: Das, Anjali
  last_name: Das
- first_name: Sarah
  full_name: Nielson, Sarah
  last_name: Nielson
- first_name: Kenneth A.
  full_name: Johnson, Kenneth A.
  last_name: Johnson
- first_name: Christopher T.
  full_name: Brown, Christopher T.
  last_name: Brown
- first_name: Cristina N.
  full_name: Butterfield, Cristina N.
  last_name: Butterfield
- first_name: Daniela S.A.
  full_name: Goltsman, Daniela S.A.
  last_name: Goltsman
- first_name: David W.
  full_name: Taylor, David W.
  last_name: Taylor
citation:
  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>
  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>
  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>.
  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.
  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>.
  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).
date_created: 2025-01-19T23:01:50Z
date_published: 2025-01-07T00:00:00Z
date_updated: 2025-07-03T11:58:22Z
day: '07'
ddc:
- '570'
department:
- _id: JaBr
doi: 10.1038/s41467-024-55573-4
external_id:
  pmid:
  - '39774105'
file:
- access_level: open_access
  checksum: 885e96690620790d5c9f188a1587b4cd
  content_type: application/pdf
  creator: dernst
  date_created: 2025-01-22T14:35:22Z
  date_updated: 2025-01-22T14:35:22Z
  file_id: '18869'
  file_name: 2025_NatureComm_Ocampo.pdf
  file_size: 5450660
  relation: main_file
  success: 1
file_date_updated: 2025-01-22T14:35:22Z
has_accepted_license: '1'
intvolume: '        16'
language:
- iso: eng
month: '01'
oa: 1
oa_version: Published Version
pmid: 1
publication: Nature Communications
publication_identifier:
  eissn:
  - 2041-1723
publication_status: published
publisher: Springer Nature
quality_controlled: '1'
scopus_import: '1'
status: public
title: DNA targeting by compact Cas9d and its resurrected ancestor
tmp:
  image: /images/cc_by.png
  legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
  name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
  short: CC BY (4.0)
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 16
year: '2025'
...
---
DOAJ_listed: '1'
_id: '15372'
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.
article_number: '3663'
article_processing_charge: Yes
article_type: original
author:
- first_name: Grace N.
  full_name: Hibshman, Grace N.
  last_name: Hibshman
- first_name: Jack Peter Kelly
  full_name: Bravo, Jack Peter Kelly
  id: 96aecfa5-8931-11ee-af30-aa6a5d6eee0e
  last_name: Bravo
  orcid: 0000-0003-0456-0753
- first_name: Matthew M.
  full_name: Hooper, Matthew M.
  last_name: Hooper
- first_name: Tyler L.
  full_name: Dangerfield, Tyler L.
  last_name: Dangerfield
- first_name: Hongshan
  full_name: Zhang, Hongshan
  last_name: Zhang
- first_name: Ilya J.
  full_name: Finkelstein, Ilya J.
  last_name: Finkelstein
- first_name: Kenneth A.
  full_name: Johnson, Kenneth A.
  last_name: Johnson
- first_name: David W.
  full_name: Taylor, David W.
  last_name: Taylor
citation:
  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>
  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>
  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>.
  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.
  ista: Hibshman GN, Bravo JPK, Hooper MM, Dangerfield TL, Zhang H, Finkelstein IJ,
    Johnson KA, Taylor DW. 2024. Unraveling the mechanisms of PAMless DNA interrogation
    by SpRY-Cas9. Nature Communications. 15, 3663.
  mla: Hibshman, Grace N., et al. “Unraveling the Mechanisms of PAMless DNA Interrogation
    by SpRY-Cas9.” <i>Nature Communications</i>, vol. 15, 3663, Springer Nature, 2024,
    doi:<a href="https://doi.org/10.1038/s41467-024-47830-3">10.1038/s41467-024-47830-3</a>.
  short: G.N. Hibshman, J.P.K. Bravo, M.M. Hooper, T.L. Dangerfield, H. Zhang, I.J.
    Finkelstein, K.A. Johnson, D.W. Taylor, Nature Communications 15 (2024).
corr_author: '1'
date_created: 2024-05-12T22:01:00Z
date_published: 2024-04-30T00:00:00Z
date_updated: 2025-05-14T09:33:21Z
day: '30'
ddc:
- '570'
department:
- _id: JaBr
doi: 10.1038/s41467-024-47830-3
external_id:
  pmid:
  - '38688943'
file:
- access_level: open_access
  checksum: 509c65919067a03ef8ad65c7192cd860
  content_type: application/pdf
  creator: dernst
  date_created: 2024-05-13T11:46:19Z
  date_updated: 2024-05-13T11:46:19Z
  file_id: '15386'
  file_name: 2024_NatureComm_Hibshman.pdf
  file_size: 7477013
  relation: main_file
  success: 1
file_date_updated: 2024-05-13T11:46:19Z
has_accepted_license: '1'
intvolume: '        15'
language:
- iso: eng
month: '04'
oa: 1
oa_version: Published Version
pmid: 1
publication: Nature Communications
publication_identifier:
  eissn:
  - 2041-1723
publication_status: published
publisher: Springer Nature
quality_controlled: '1'
scopus_import: '1'
status: public
title: Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9
tmp:
  image: /images/cc_by.png
  legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
  name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
  short: CC BY (4.0)
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 15
year: '2024'
...
---
OA_place: repository
OA_type: green
_id: '17442'
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"
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.).
article_processing_charge: No
article_type: original
author:
- first_name: Jack Peter Kelly
  full_name: Bravo, Jack Peter Kelly
  id: 96aecfa5-8931-11ee-af30-aa6a5d6eee0e
  last_name: Bravo
  orcid: 0000-0003-0456-0753
- first_name: Delisa A.
  full_name: Ramos, Delisa A.
  last_name: Ramos
- first_name: Rodrigo
  full_name: Fregoso Ocampo, Rodrigo
  last_name: Fregoso Ocampo
- first_name: Caiden
  full_name: Ingram, Caiden
  last_name: Ingram
- first_name: David W.
  full_name: Taylor, David W.
  last_name: Taylor
citation:
  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>
  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>
  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>.
  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.
  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.
  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>.
  short: J.P.K. Bravo, D.A. Ramos, R. Fregoso Ocampo, C. Ingram, D.W. Taylor, Nature
    630 (2024) 961–967.
corr_author: '1'
date_created: 2024-08-19T09:41:18Z
date_published: 2024-06-27T00:00:00Z
date_updated: 2025-06-24T12:47:21Z
day: '27'
department:
- _id: JaBr
doi: 10.1038/s41586-024-07515-9
external_id:
  pmid:
  - '38740055'
intvolume: '       630'
issue: '8018'
language:
- iso: eng
main_file_link:
- open_access: '1'
  url: https://pmc.ncbi.nlm.nih.gov/articles/PMC11649018/
month: '06'
oa: 1
oa_version: Submitted Version
page: 961-967
pmid: 1
publication: Nature
publication_identifier:
  eissn:
  - 1476-4687
  issn:
  - 0028-0836
publication_status: published
publisher: Springer Nature
quality_controlled: '1'
scopus_import: '1'
status: public
title: Plasmid targeting and destruction by the DdmDE bacterial defence system
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 630
year: '2024'
...
---
OA_place: publisher
OA_type: free access
_id: '17494'
acknowledgement: I would like to thank K Kiernan for insightful comments and feedback.
  J P K Bravo is supported by IST Austria.
article_processing_charge: No
article_type: letter_note
author:
- first_name: Jack Peter Kelly
  full_name: Bravo, Jack Peter Kelly
  id: 96aecfa5-8931-11ee-af30-aa6a5d6eee0e
  last_name: Bravo
  orcid: 0000-0003-0456-0753
citation:
  ama: 'Bravo JPK. Anti-plasmid immunity: A key to pathogen success? <i>Future Microbiology</i>.
    2024;19(15):1269-1272. doi:<a href="https://doi.org/10.1080/17460913.2024.2389720">10.1080/17460913.2024.2389720</a>'
  apa: 'Bravo, J. P. K. (2024). Anti-plasmid immunity: A key to pathogen success?
    <i>Future Microbiology</i>. Taylor &#38; Francis. <a href="https://doi.org/10.1080/17460913.2024.2389720">https://doi.org/10.1080/17460913.2024.2389720</a>'
  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>.'
  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.'
  ista: 'Bravo JPK. 2024. Anti-plasmid immunity: A key to pathogen success? Future
    Microbiology. 19(15), 1269–1272.'
  mla: 'Bravo, Jack Peter Kelly. “Anti-Plasmid Immunity: A Key to Pathogen Success?”
    <i>Future Microbiology</i>, vol. 19, no. 15, Taylor &#38; Francis, 2024, pp. 1269–72,
    doi:<a href="https://doi.org/10.1080/17460913.2024.2389720">10.1080/17460913.2024.2389720</a>.'
  short: J.P.K. Bravo, Future Microbiology 19 (2024) 1269–1272.
corr_author: '1'
date_created: 2024-09-05T07:32:00Z
date_published: 2024-10-01T00:00:00Z
date_updated: 2025-09-08T09:03:00Z
day: '01'
department:
- _id: JaBr
doi: 10.1080/17460913.2024.2389720
external_id:
  isi:
  - '001306115400001'
  pmid:
  - '39230568'
has_accepted_license: '1'
intvolume: '        19'
isi: 1
issue: '15'
language:
- iso: eng
main_file_link:
- open_access: '1'
  url: https://doi.org/10.1080/17460913.2024.2389720
month: '10'
oa: 1
oa_version: Published Version
page: 1269-1272
pmid: 1
publication: Future Microbiology
publication_identifier:
  eissn:
  - 1746-0921
  issn:
  - 1746-0913
publication_status: published
publisher: Taylor & Francis
quality_controlled: '1'
scopus_import: '1'
status: public
title: 'Anti-plasmid immunity: A key to pathogen success?'
type: journal_article
user_id: 317138e5-6ab7-11ef-aa6d-ffef3953e345
volume: 19
year: '2024'
...