Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Precise virulence inactivation using a CRISPR-associated transposase for combating Enterobacteriaceae gut pathogens

Nature Biomedical Engineering volume 9, pages 2017–2027 (2025)Cite this article

Subjects

Abstract

Targeted gene manipulation in a complex microbial community is an enabling technology for precise microbiome editing. Here we introduce BACTRINS, an in situ microbiome engineering platform designed for efficient and precise genomic insertion of a desired payload and simultaneous knockout of target genes. This system leverages conjugation-mediated delivery of CRISPR-associated transposases to achieve RNA-guided genomic integration, allowing precise insertion of a therapeutic payload while neutralizing pathogen virulence without causing cell death. When applied against an Enterobacteriaceae Shiga toxin-producing pathogen in the gut, this system delivers a CRISPR-associated transposase by bacterial conjugation for site-specific inactivation of the Shiga toxin gene and integration of a nanobody therapeutic payload to disrupt pathogen attachment. A single dose of this therapy results in high-efficiency Shiga gene inactivation and improved survival in a murine infection model of Shiga-producing pathogen. This work establishes a new type of live bacterial therapeutic capable of reducing gut infections by transforming toxigenic pathogens into commensal protectors.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overview of BACTRINS system for bacterial virulence reduction in situ.
Fig. 2: In vitro validation of the BACTRINS system.
Fig. 3: In vivo characterization of the BACTRINS system.
Fig. 4: Optimized BACTRINS system incorporating a therapeutic nanobody payload.

Similar content being viewed by others

Data availability

All raw experimental data for the figures are available in the source data provided with this paper. Raw data of the Supplementary Information are available upon request.

References

  1. Mayorga-Ramos, A., Zúñiga-Miranda, J., Carrera-Pacheco, S. E., Barba-Ostria, C. & Guamán, L. P. CRISPR-Cas-based antimicrobials: design, challenges, and bacterial mechanisms of resistance. ACS Infect. Dis. 9, 1283–1302 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Song, Z. et al. Pathogen-specific bactericidal method mediated by conjugative delivery of CRISPR-Cas13a targeting bacterial endogenous transcripts. Microbiol. Spectr. 10, e01300-22 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Neil, K. et al. High‐efficiency delivery of CRISPR‐Cas9 by engineered probiotics enables precise microbiome editing. Mol. Syst. Biol. 17, e10335 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Sheng, H. et al. Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance. PLoS ONE 18, e0291520 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Hamilton, T. A. et al. Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing. Nat. Commun. 10, 4544 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Reuter, A. et al. Targeted-antibacterial-plasmids (TAPs) combining conjugation and CRISPR/Cas systems achieve strain-specific antibacterial activity. Nucleic Acids Res. 49, 3584–3598 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Gencay, Y. E. et al. Engineered phage with antibacterial CRISPR–Cas selectively reduce E. coli burden in mice. Nat. Biotechnol. 42, 265–274 (2024).

    Article  PubMed  CAS  Google Scholar 

  8. Citorik, R. J., Mimee, M. & Lu, T. K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat. Biotechnol. 32, 1141–1145 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Bikard, D. et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Selle, K. et al. In vivo targeting of Clostridioides difficile using phage-delivered CRISPR-Cas3 antimicrobials. mBio 11, e00019-20 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Kim, P. et al. Safety, pharmacokinetics, and pharmacodynamics of LBP-EC01, a CRISPR-Cas3-enhanced bacteriophage cocktail, in uncomplicated urinary tract infections due to Escherichia coli (ELIMINATE): the randomised, open-label, first part of a two-part phase 2 trial. Lancet Infect. Dis. https://doi.org/10.1016/S1473-3099(24)00424-9 (2024).

  12. Kiga, K. et al. Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria. Nat. Commun. 11, 2934 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Jiang, W. et al. Dealing with the evolutionary downside of CRISPR immunity: bacteria and beneficial plasmids. PLoS Genet. 9, e1003844 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Khalil, I. A. et al. Morbidity and mortality due to Shigella and enterotoxigenic Escherichia coli diarrhoea: the Global Burden of Disease Study 1990–2016. Lancet Infect. Dis. 18, 1229–1240 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Scallan, E., Griffin, P. M., Angulo, F. J., Tauxe, R. V. & Hoekstra, R. M. Foodborne illness acquired in the United States—unspecified agents. Emerg. Infect. Dis. 17, 16–22 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Etcheverría, A. I. & Padola, N. L. Shiga toxin-producing Escherichia coli: factors involved in virulence and cattle colonization. Virulence 4, 366–372 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Paton, J. C. & Paton, A. W. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11, 450–479 (1998).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Naidoo, N. & Zishiri, O. T. Comparative genomics analysis and characterization of Shiga toxin-producing Escherichia coli O157:H7 strains reveal virulence genes, resistance genes, prophages and plasmids. BMC Genomics 24, 791 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Hall, G., Kurosawa, S. & Stearns-Kurosawa, D. Shiga toxin therapeutics: beyond neutralization. Toxins 9, 291 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Mühlen, S. & Dersch, P. Treatment strategies for infections with Shiga toxin-producing Escherichia coli. Front. Cell. Infect. Microbiol. 10, 169 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Freedman, S. B. et al. Hyperhydration to improve kidney outcomes in children with Shiga toxin-producing E. coli infection: a multinational embedded cluster crossover randomized trial (the HIKO STEC trial). Trials 24, 359 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Tarr, P. I. & Freedman, S. B. Why antibiotics should not be used to treat Shiga toxin-producing Escherichia coli infections. Curr. Opin. Gastroenterol. 38, 30–38 (2022).

    Article  PubMed  CAS  Google Scholar 

  23. Bielaszewska, M. et al. Effects of antibiotics on Shiga toxin 2 production and bacteriophage induction by epidemic Escherichia coli O104:H4 strain. Antimicrob. Agents Chemother. 56, 3277–3282 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Ruano-Gallego, D. & Fernández, L. Á. in Shiga Toxin-Producing E. coli (eds Schüller, S. & Bielaszewska, M.) 253–272 (Springer, 2021).

  25. Cheng, L., Henderson, T., Patfield, S., Stanker, L. & He, X. Mouse in vivo neutralization of Escherichia coli Shiga toxin 2 with monoclonal antibodies. Toxins 5, 1845–1858 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Schmidt, N. et al. Decreased STEC shedding by cattle following passive and active vaccination based on recombinant Escherichia coli Shiga toxoids. Vet. Res. 49, 28 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Sreerohini, S., Balakrishna, K. & Parida, M. Oral immunization of mice with Lactococcus lactis expressing Shiga toxin truncate confers enhanced protection against Shiga toxins of Escherichia coli O157:H7 and Shigella dysenteriae. APMIS 127, 671–680 (2019).

    Article  PubMed  CAS  Google Scholar 

  28. Cai, K., Tu, W., Liu, Y., Li, T. & Wang, H. Novel fusion antigen displayed-bacterial ghosts vaccine candidate against infection of Escherichia coli O157:H7. Sci. Rep. 5, 17479 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Kim, B. et al. Phage-derived antibacterials: harnessing the simplicity, plasticity, and diversity of phages. Viruses 11, 268 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Rund, S. A., Rohde, H., Sonnenborn, U. & Oelschlaeger, T. A. Antagonistic effects of probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4 and O157:H7. Int. J. Med. Microbiol. 303, 1–8 (2013).

    Article  PubMed  CAS  Google Scholar 

  31. Safwat Mohamed, D., Farouk Ahmed, E., Mohamed Mahmoud, A., Abd El-Baky, R. M. & John, J. Isolation and evaluation of cocktail phages for the control of multidrug-resistant Escherichia coli serotype O104: H4 and E. coli O157: H7 isolates causing diarrhea. FEMS Microbiol. Lett. https://doi.org/10.1093/femsle/fnx275 (2018).

  32. Jia, M., Geornaras, I., Belk, K. E. & Yang, H. Sequence-specific removal of Shiga toxin-producing Escherichia coli using the CRISPR-Cas9 system. Meat Muscle Biol. https://doi.org/10.22175/rmc2017.116 (2019).

  33. Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S. & Sternberg, S. H. Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature 571, 219–225 (2019).

    Article  PubMed  CAS  Google Scholar 

  34. Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48–53 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Vo, P. L. H. et al. CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nat. Biotechnol. 39, 480–489 (2021).

  36. Rubin, B. E. et al. Species- and site-specific genome editing in complex bacterial communities. Nat. Microbiol. 7, 34–47 (2022).

    Article  PubMed  CAS  Google Scholar 

  37. Ronda, C., Chen, S. P., Cabral, V., Yaung, S. J. & Wang, H. H. Metagenomic engineering of the mammalian gut microbiome in situ. Nat. Methods 16, 167–170 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Li, C. et al. Efficient CRISPR-Cas9 based cytosine base editors for phytopathogenic bacteria. Commun. Biol. 6, 56 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Zheng, K. et al. Highly efficient base editing in bacteria using a Cas9-cytidine deaminase fusion. Commun. Biol. 1, 32 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Farzadfard, F. & Lu, T. K. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations. Science 346, 1256272 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Schubert, M. G. et al. High-throughput functional variant screens via in vivo production of single-stranded DNA. Proc. Natl Acad. Sci. USA 118, e2018181118 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Beers, K. et al. Comparison of the RAPID-B® flow cytometer and the BAX® system for the detection of non-O157 Shiga toxin-producing Escherichia coli (STEC) in beef products. Food Control 50, 72–75 (2015).

    Article  CAS  Google Scholar 

  44. Beaulaurier, J. et al. Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation. Nat. Biotechnol. 36, 61–69 (2018).

    Article  PubMed  CAS  Google Scholar 

  45. OPINION 26 Designation of neotype strains (cultures) of type species of the bacterial genera Salmonella, Shigella, Arizona, Escherichia, Citrobacter and Proteus of the Family Enterobacteriaceae. Int. J. Syst. Evol. Microbiol. 13, 35–36 (1963).

  46. Shin, H. H., Seo, J. H., Kim, C. S., Hwang, B. H. & Cha, H. J. Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae. Biosens. Bioelectron. 79, 398–405 (2016).

    Article  PubMed  CAS  Google Scholar 

  47. Wells, J. G. et al. Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J. Clin. Microbiol. 18, 512–520 (1983).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Dou, L. et al. ‘Three-To-One’ multi-functional nanocomposite-based lateral flow immunoassay for label-free and dual-readout detection of pathogenic bacteria. Biosens. Bioelectron. 204, 114093 (2022).

    Article  PubMed  CAS  Google Scholar 

  49. Strockbine, N. A. et al. Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities. Infect. Immun. 53, 135–140 (1986).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Kudva, I. T., Hatfield, P. G. & Hovde, C. J. Escherichia coli O157:H7 in microbial flora of sheep. J. Clin. Microbiol. 34, 431–433 (1996).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Marques, L. R. et al. Production of Shiga-like toxin by Escherichia coli. J. Infect. Dis. 154, 338–341 (1986).

    Article  PubMed  CAS  Google Scholar 

  52. Plunkett, G., Rose, D. J., Durfee, T. J. & Blattner, F. R. Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-gene product. J. Bacteriol. 181, 1767–1778 (1999).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Shaw, L. P., Rocha, E. P. C. & MacLean, R. C. Restriction-modification systems have shaped the evolution and distribution of plasmids across bacteria. Nucleic Acids Res. 51, 6806–6818 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Westra, E. R. et al. CRISPR-Cas systems preferentially target the leading regions of MOBF conjugative plasmids. RNA Biol. 10, 749–761 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Novick, R. P. Plasmid incompatibility. Microbiol. Rev. 51, 381–395 (1987).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Allard, N., Collette, A., Paquette, J., Rodrigue, S. & Côté, J.-P. Systematic investigation of recipient cell genetic requirements reveals important surface receptors for conjugative transfer of IncI2 plasmids. Commun. Biol. 6, 1172 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Neil, K., Allard, N., Grenier, F., Burrus, V. & Rodrigue, S. Highly efficient gene transfer in the mouse gut microbiota is enabled by the Incl2 conjugative plasmid TP114. Commun. Biol. 3, 523 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Kennedy, E. A., King, K. Y. & Baldridge, M. T. Mouse microbiota models: comparing germ-free mice and antibiotics treatment as tools for modifying gut bacteria. Front. Physiol. 9, 1534 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Thorpe, C. M., Ritchie, J. M. & Acheson, D. W. K. in Escherichia coli (ed. Donnenberg, M. S.) 119–154 (Elsevier, 2002).

  60. Jeon, B., Saisom, T., Sasipreeyajan, J. & Luangtongkum, T. Live-attenuated oral vaccines to reduce Campylobacter colonization in poultry. Vaccines 10, 685 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Phillips, A. D. & Frankel, G. Intimin‐mediated tissue specificity in enteropathogenic Escherichia coli interaction with human intestinal organ cultures. J. Infect. Dis. 181, 1496–1500 (2000).

    Article  PubMed  CAS  Google Scholar 

  62. Lewis, S. B., Cook, V., Tighe, R. & Schüller, S. Enterohemorrhagic Escherichia coli colonization of human colonic epithelium in vitro and ex vivo. Infect. Immun. 83, 942–949 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Ruano-Gallego, D. et al. A nanobody targeting the translocated intimin receptor inhibits the attachment of enterohemorrhagic E. coli to human colonic mucosa. PLoS Pathog. 15, e1008031 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Han, S. et al. Novel signal peptides improve the secretion of recombinant Staphylococcus aureus alpha toxinH35L in Escherichia coli. AMB Express 7, 93 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Tamma, P. D. et al. Safety and microbiological activity of phage therapy in persons with cystic fibrosis colonized with Pseudomonas aeruginosa: study protocol for a phase 1b/2, multicenter, randomized, double-blind, placebo-controlled trial. Trials 23, 1057 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. Nethery, M. A., Hidalgo-Cantabrana, C., Roberts, A. & Barrangou, R. CRISPR-based engineering of phages for in situ bacterial base editing. Proc. Natl Acad. Sci. USA 119, e2206744119 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Zhou, W., Chow, K., Fleming, E. & Oh, J. Selective colonization ability of human fecal microbes in different mouse gut environments. ISME J. 13, 805–823 (2019).

    Article  PubMed  CAS  Google Scholar 

  68. Paton, A. W. et al. Molecular microbiological investigation of an outbreak of hemolytic-uremic syndrome caused by dry fermented sausage contaminated with Shiga-like toxin-producing Escherichia coli. J. Clin. Microbiol. 34, 1622–1627 (1996).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  69. Spinale, J. M., Ruebner, R. L., Copelovitch, L. & Kaplan, B. S. Long-term outcomes of Shiga toxin hemolytic uremic syndrome. Pediatr. Nephrol. 28, 2097–2105 (2013).

    Article  PubMed  Google Scholar 

  70. Lu, Z. et al. Nanobody-based bispecific neutralizer for Shiga toxin-producing E. coli. ACS Infect. Dis. 8, 321–329 (2022).

    Article  PubMed  CAS  Google Scholar 

  71. Klompe, S. E. et al. Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons. Mol. Cell 82, 616–628.e5 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  72. Harimoto, T. et al. A programmable encapsulation system improves delivery of therapeutic bacteria in mice. Nat. Biotechnol. 40, 1259–1269 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  73. Razavi, S., Janfaza, S., Tasnim, N., Gibson, D. L. & Hoorfar, M. Nanomaterial-based encapsulation for controlled gastrointestinal delivery of viable probiotic bacteria. Nanoscale Adv. 3, 2699–2709 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  74. Lam, K. N. et al. Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome. Cell Rep. 37, 109930 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  75. Isabella, V. M. et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat. Biotechnol. 36, 857–864 (2018).

    Article  PubMed  CAS  Google Scholar 

  76. Pleguezuelos-Manzano, C. et al. Mutational signature in colorectal cancer caused by genotoxic pks+ E. coli. Nature 580, 269–273 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  77. Kalantari, A. et al. Robust performance of a live bacterial therapeutic chassis lacking the colibactin gene cluster. PLoS ONE 18, e0280499 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  78. Koch, L. CRISPR editing within microbial communities. Nat. Rev. Genet. 23, 72 (2022).

    PubMed  Google Scholar 

  79. Pantoja Angles, A., Valle-Pérez, A. U., Hauser, C. & Mahfouz, M. M. Microbial biocontainment systems for clinical, agricultural, and industrial applications. Front. Bioeng. Biotechnol. 10, 830200 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  80. Foo, J. L., Ling, H., Lee, Y. S. & Chang, M. W. Microbiome engineering: current applications and its future. Biotechnol. J. 12, 1600099 (2017).

    Article  Google Scholar 

  81. Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinformatics 10, 421 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  82. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  83. Simon, R., Priefer, U. & Pühler, A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Nat. Biotechnol. 1, 784–791 (1983).

    Article  CAS  Google Scholar 

  84. Neil, K., Allard, N., Jordan, D. & Rodrigue, S. Assembly of large mobilizable genetic cargo by double recombinase operated insertion of DNA (DROID). Plasmid 104, 102419 (2019).

    Article  PubMed  CAS  Google Scholar 

  85. Datta, S., Costantino, N. & Court, D. L. A set of recombineering plasmids for Gram-negative bacteria. Gene 379, 109–115 (2006).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank members of the Wang laboratory for support and comments on the paper, including G. Urtecho and C. Mavros. S.H.S. was supported by NIH grants DP2HG011650, R21AI68976 and R01EB031935; a Pew Biomedical Scholarship; a Sloan Research Fellowship; an Irma T. Hirschl Career Scientist Award; the Howard Hughes Medical Institute; and a generous startup package from the Columbia University Irving Medical Center Dean’s Office and the Vagelos Precision Medicine Fund. H.H.W. acknowledges funding support from the NSF (MCB-2025515), NIH (1R01EB031935, 2R01AI132403, 1R01DK118044, 1R21AI146817, 5P30DK132710), DOD (S-168-4X5-001), Burroughs Wellcome Fund (1016691), Irma T. Hirschl Trust, and Schaefer Research Award. C.R. was supported for part of this project as Junior Fellow by the Simons Society of Fellows (527896) and she is now supported in part by Lyda Hill Philanthropies, Acton Family Giving, the Valhalla Foundation, Hastings/Quillin Fund - an advised fund of the Silicon Valley Community Foundation, the CH Foundation, Laura and Gary Lauder and Family, the Sea Grape Foundation, the Emerson Collective, Mike Schroepfer and Erin Hoffman Family Fund - an advised fund of Silicon Valley Community Foundation, and the Anne Wojcicki Foundation through The Audacious Project at the Innovative Genomics Institute.

Author information

Author notes

  1. These authors contributed equally: Carlotta Ronda, Tyler Perdue.

Authors and Affiliations

  1. Department of System Biology, Columbia University, New York, NY, USA

    Carlotta Ronda, Tyler Perdue, Logan Schwanz, Diego Rivera Gelsinger, Leonie Brockmann, Andrew Kaufman, Yiming Huang & Harris H. Wang

  2. Innovative Genomics Institute, University California Berkeley, Berkeley, CA, USA

    Carlotta Ronda

  3. Department of Biological Sciences, Columbia University, New York, NY, USA

    Tyler Perdue

  4. Department of Pathology and Cell Biology, Columbia University, New York, NY, USA

    Logan Schwanz & Harris H. Wang

  5. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Diego Rivera Gelsinger & Samuel H. Sternberg

  6. Howard Hughes Medical Institute, Columbia University, New York, NY, USA

    Samuel H. Sternberg

  7. Department of Biomedical Engineering, Columbia University, New York, USA

    Harris H. Wang

Authors
  1. Carlotta Ronda
    View author publications

    Search author on:PubMed Google Scholar

  2. Tyler Perdue
    View author publications

    Search author on:PubMed Google Scholar

  3. Logan Schwanz
    View author publications

    Search author on:PubMed Google Scholar

  4. Diego Rivera Gelsinger
    View author publications

    Search author on:PubMed Google Scholar

  5. Leonie Brockmann
    View author publications

    Search author on:PubMed Google Scholar

  6. Andrew Kaufman
    View author publications

    Search author on:PubMed Google Scholar

  7. Yiming Huang
    View author publications

    Search author on:PubMed Google Scholar

  8. Samuel H. Sternberg
    View author publications

    Search author on:PubMed Google Scholar

  9. Harris H. Wang
    View author publications

    Search author on:PubMed Google Scholar

Contributions

C.R. and H.H.W. developed the initial concept with inputs from S.H.S.; T.P., C.R., L.S. and D.R.G. performed in vitro experiments; C.R., T.P., A.K. and L.B. performed animal experiments; T.P., C.R. and Y.H. analysed the data. T.P. and H.H.W. wrote the paper. All other authors discussed results and approved the paper.

Corresponding author

Correspondence to Harris H. Wang.

Ethics declarations

Competing interests

Patent application (patent application number 18/460,286; 1 September 2023, New York, NY, USA) describing the CAST and gene delivery technologies has been filed by Columbia University. S.H.S. is a co-founder and scientific adviser to Dahlia Biosciences, a scientific adviser to CrisprBits and Prime Medicine, and an equity holder in Dahlia Biosciences and CrisprBits. H.H.W. is a scientific advisor of SNIPR Biome, Kingdom Supercultures, Fitbiomics, VecX Biomedicines and Genus PLC, and a scientific co-founder of Aclid and Foli Bio, all of which are not involved in the study.

Peer review

Peer review information

Nature Biomedical Engineering thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ronda, C., Perdue, T., Schwanz, L. et al. Precise virulence inactivation using a CRISPR-associated transposase for combating Enterobacteriaceae gut pathogens. Nat. Biomed. Eng 9, 2017–2027 (2025). https://doi.org/10.1038/s41551-025-01453-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41551-025-01453-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research