Genome editing of phylogenetically distinct bacteria using cross-species retron-mediated recombineering
Arroyo-Olarte, R. D., Bravo Rodríguez, R. & Morales-Ríos, E. Genome editing in bacteria: CRISPR–Cas and beyond. Microorganisms 9, 844 (2021).
Google Scholar
Vento, J. M., Crook, N. & Beisel, C. L. Barriers to genome editing with CRISPR in bacteria. J. Ind. Microbiol. Biotechnol. 46, 1327–1341 (2019).
Google Scholar
Wannier, T. M. et al. Recombineering and MAGE. Nat. Rev. Methods Primer 1, 7 (2021).
Google Scholar
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).
Google Scholar
Yu, D. et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl Acad. Sci. USA 97, 5978–5983 (2000).
Google Scholar
Ellis, H. M., Yu, D., DiTizio, T. & Court, D. L. High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl Acad. Sci. USA 98, 6742–6746 (2001).
Google Scholar
Mosberg, J. A., Lajoie, M. J. & Church, G. M. Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186, 791–799 (2010).
Google Scholar
Wannier, T. M. et al. Improved bacterial recombineering by parallelized protein discovery. Proc. Natl Acad. Sci. USA 117, 13689–13698 (2020).
Google Scholar
Filsinger, G. T. et al. Characterizing the portability of phage-encoded homologous recombination proteins. Nat. Chem. Biol. 17, 394–402 (2021).
Google Scholar
Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).
Google Scholar
Ronda, C., Pedersen, L. E., Sommer, M. O. A. & Nielsen, A. T. CRMAGE: CRISPR optimized MAGE recombineering. Sci. Rep. 6, 19452 (2016).
Google Scholar
Corts, A., Thomason, L. C., Costantino, N. & Court, D. L. Recombineering in non‐model bacteria. Curr. Protoc. 2, e605 (2022).
Google Scholar
Isaacs, F. J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348–353 (2011).
Google Scholar
Millman, A. et al. Bacterial retrons function in anti-phage defense. Cell 183, 1551–1561.e12 (2020).
Google Scholar
Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084 (2020).
Google Scholar
Mestre, M. R., González-Delgado, A., Gutiérrez-Rus, L. I., Martínez-Abarca, F. & Toro, N. Systematic prediction of genes functionally associated with bacterial retrons and classification of the encoded tripartite systems. Nucleic Acids Res. 48, 12632–12647 (2020).
Google Scholar
Inouye, S., Hsu, M.-Y., Eagle, S. & Inouye, M. Reverse transcriptase associated with the biosynthesis of the branched RNA-linked msDNA in Myxococcus xanthus. Cell 56, 709–717 (1989).
Google Scholar
Lim, D. & Maas, W. K. Reverse transcriptase-dependent synthesis of a covalently linked, branched DNA–RNA compound in E. coli B. Cell 56, 891–904 (1989).
Bobonis, J. et al. Bacterial retrons encode phage-defending tripartite toxin–antitoxin systems. Nature 609, 144–150 (2022).
Google Scholar
Palka, C., Fishman, C. B., Bhattarai-Kline, S., Myers, S. A. & Shipman, S. L. Retron reverse transcriptase termination and phage defense are dependent on host RNase H1. Nucleic Acids Res. 50, 3490–3504 (2022).
Google Scholar
Carabias, A. et al. Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages. Mol. Cell 84, 2185–2202.e12 (2024).
Google Scholar
Farzadfard, F. & Lu, T. K. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations. Science 346, 1256272 (2014).
Google Scholar
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).
Google Scholar
Lopez, S. C., Crawford, K. D., Lear, S. K., Bhattarai-Kline, S. & Shipman, S. L. Precise genome editing across kingdoms of life using retron-derived DNA. Nat. Chem. Biol. 18, 199–206 (2022).
Google Scholar
Fishman, C. B. et al. Continuous multiplexed phage genome editing using recombitrons. Nat. Biotechnol. 43, 1299–1310 (2025).
Google Scholar
Liu, W. et al. Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli. Nucleic Acids Res. 51, 8293–8307 (2023).
Google Scholar
González-Delgado, A., Lopez, S. C., Rojas-Montero, M., Fishman, C. B. & Shipman, S. L. Simultaneous multi-site editing of individual genomes using retron arrays. Nat. Chem. Biol. 20, 1482–1492 (2024).
Google Scholar
Simon, A. J., Morrow, B. R. & Ellington, A. D. Retroelement-based genome editing and evolution. ACS Synth. Biol. 7, 2600–2611 (2018).
Google Scholar
Liu, W. et al. Intracellularly synthesized ssDNA for continuous genome engineering. Trends Biotechnol. 43, 1356–1370 (2025).
Google Scholar
Ni, Y. et al. Reducing competition between msd and genomic DNA improves retron editing efficiency. EMBO Rep. 25, 5316–5330 (2024).
Google Scholar
Khan, A. G. et al. An experimental census of retrons for DNA production and genome editing. Nat. Biotechnol. 43, 914–922 (2025).
Google Scholar
Jiang, P. et al. Pathway redesign for deoxyviolacein biosynthesis in Citrobacter freundii and characterization of this pigment. Appl. Microbiol. Biotechnol. 94, 1521–1532 (2012).
Google Scholar
Metsoviti, M., Zeng, A.-P., Koutinas, A. A. & Papanikolaou, S. Enhanced 1,3-propanediol production by a newly isolated Citrobacter freundii strain cultivated on biodiesel-derived waste glycerol through sterile and non-sterile bioprocesses. J. Biotechnol. 163, 408–418 (2013).
Google Scholar
Ramos-Vivas, J. et al. Adherence to human colon cells by multidrug resistant Enterobacterales strains isolated from solid organ transplant recipients with a focus on Citrobacter freundii. Front. Cell. Infect. Microbiol. 10, 447 (2020).
Google Scholar
Chen, X., Zhou, Y. & Feng, Y. Citrobacter freundii induces sepsis with new-onset status seizure in an adult: a case report and literature review. Medicine (Baltimore) 102, e32549 (2023).
Google Scholar
Tommasi, R., Brown, D. G., Walkup, G. K., Manchester, J. I. & Miller, A. A. ESKAPEing the labyrinth of antibacterial discovery. Nat. Rev. Drug Discov. 14, 529–542 (2015).
Google Scholar
Tang, M., Kong, X., Hao, J. & Liu, J. Epidemiological characteristics and formation mechanisms of multidrug-resistant hypervirulent Klebsiella pneumoniae. Front. Microbiol. 11, 581543 (2020).
Google Scholar
Tesson, F. et al. Systematic and quantitative view of the antiviral arsenal of prokaryotes. Nat. Commun. 13, 2561 (2022).
Google Scholar
Bonn, W. G. & Zwet, T. V. D. in Fire blight: The Disease and its Causative Agent, Erwinia amylovora (ed. Vanneste, J. L.) 37–53 (CABI Publishing, 2000); https://doi.org/10.1079/9780851992945.0037
Van Der Zwet, T., Orolaza-Halbrendt, N. & Zeller, W. Fire Blight: History, Biology, and Management (The American Phytopathological Society, 2016); https://doi.org/10.1094/9780890544839
Weinstock, M. T., Hesek, E. D., Wilson, C. M. & Gibson, D. G. Vibrio natriegens as a fast-growing host for molecular biology. Nat. Methods 13, 849–851 (2016).
Google Scholar
Janda, J. M. & Abbott, S. L. The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin. Microbiol. Rev. 23, 35–73 (2010).
Google Scholar
Schwartz, K. et al. Emerging Aeromonas spp. infections in Europe: characterization of human clinical isolates from German patients. Front. Microbiol. 15, 1498180 (2024).
Google Scholar
Hau, H. H. & Gralnick, J. A. Ecology and biotechnology of the genus Shewanella. Annu. Rev. Microbiol. 61, 237–258 (2007).
Google Scholar
Munoz-Cupa, C., Hu, Y., Xu, C. & Bassi, A. An overview of microbial fuel cell usage in wastewater treatment, resource recovery and energy production. Sci. Total Environ. 754, 142429 (2021).
Google Scholar
Yang, E. et al. A review on self-sustainable microbial electrolysis cells for electro-biohydrogen production via coupling with carbon-neutral renewable energy technologies. Bioresour. Technol. 320, 124363 (2021).
Google Scholar
Corts, A. D., Thomason, L. C., Gill, R. T. & Gralnick, J. A. Efficient and precise genome editing in Shewanella with recombineering and CRISPR/Cas9-mediated counter-selection. ACS Synth. Biol. 8, 1877–1889 (2019).
Google Scholar
Swingle, B., Bao, Z., Markel, E., Chambers, A. & Cartinhour, S. Recombineering using RecTE from Pseudomonas syringae. Appl. Environ. Microbiol. 76, 4960–4968 (2010).
Google Scholar
Aparicio, T., Nyerges, A., Martínez-García, E. & De Lorenzo, V. High-efficiency multi-site genomic editing of Pseudomonas putida through thermoinducible ssDNA recombineering. iScience 23, 100946 (2020).
Google Scholar
Angeletti, S. et al. Multi-drug resistant Pseudomonas aeruginosa nosocomial strains: molecular epidemiology and evolution. Microb. Pathog. 123, 233–241 (2018).
Google Scholar
Sriramulu, D. (ed) Pseudomonas aeruginosa: An Armory Within (IntechOpen, 2019).
Loeschcke, A. & Thies, S. Pseudomonas putida—a versatile host for the production of natural products. Appl. Microbiol. Biotechnol. 99, 6197–6214 (2015).
Google Scholar
Martínez-García, E. & De Lorenzo, V. Molecular tools and emerging strategies for deep genetic/genomic refactoring of Pseudomonas. Curr. Opin. Biotechnol. 47, 120–132 (2017).
Google Scholar
Xin, X.-F., Kvitko, B. & He, S. Y. Pseudomonas syringae: what it takes to be a pathogen. Nat. Rev. Microbiol. 16, 316–328 (2018).
Google Scholar
Elliott, K. T. & Neidle, E. L. Acinetobacter baylyi ADP1: transforming the choice of model organism. IUBMB Life 63, 1075–1080 (2011).
Google Scholar
Santala, S. & Santala, V. Acinetobacter baylyi ADP1—naturally competent for synthetic biology. Essays Biochem. 65, 309–318 (2021).
Google Scholar
Asin-Garcia, E. et al. Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species. Nucleic Acids Res. 51, 12522–12536 (2023).
Google Scholar
Biggs, B. W. et al. Development of a genetic toolset for the highly engineerable and metabolically versatile Acinetobacter baylyi ADP1. Nucleic Acids Res. 48, 5169–5182 (2020).
Google Scholar
Bryksin, A. V. & Matsumura, I. Rational design of a plasmid origin that replicates efficiently in both Gram-positive and Gram-negative bacteria. PLoS ONE 5, e13244 (2010).
Google Scholar
Mu, Q., Tavella, V. J. & Luo, X. M. Role of Lactobacillus reuteri in human health and diseases. Front. Microbiol. 9, 757 (2018).
Google Scholar
Yu, Z. et al. The role of potential probiotic strains Lactobacillus reuteri in various intestinal diseases: new roles for an old player. Front. Microbiol. 14, 1095555 (2023).
Google Scholar
Wertheim, H. F. L., Nghia, H. D. T., Taylor, W. & Schultsz, C. Streptococcus suis: an emerging human pathogen. Clin. Infect. Dis. 48, 617–625 (2009).
Google Scholar
Fredriksen, S. et al. Streptococcus suis infection on European farms is associated with an altered tonsil microbiome and resistome. Microb. Genomics 10, 001334 (2024).
Google Scholar
Sparks, I. L., Derbyshire, K. M., Jacobs, W. R. & Morita, Y. S. Mycobacterium smegmatis: the vanguard of mycobacterial research. J. Bacteriol. 205, e0033722 (2023).
Google Scholar
Mayslich, C., Grange, P. A. & Dupin, N. Cutibacterium acnes as an opportunistic pathogen: an update of its virulence-associated factors. Microorganisms 9, 303 (2021).
Google Scholar
Cros, M. P. et al. New insights into the role of Cutibacterium acnes-derived extracellular vesicles in inflammatory skin disorders. Sci. Rep. 13, 16058 (2023).
Google Scholar
Van Pijkeren, J.-P. & Britton, R. A. High efficiency recombineering in lactic acid bacteria. Nucleic Acids Res. 40, e76 (2012).
Google Scholar
Risøen, P. A., Brurberg, M. B., Eijsink, V. G. H. & Nes, I. F. Functional analysis of promoters involved in quorum sensing‐based regulation of bacteriocin production in Lactobacillus. Mol. Microbiol. 37, 619–628 (2000).
Google Scholar
Pavan, S. et al. Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects. Appl. Environ. Microbiol. 66, 4427–4432 (2000).
Google Scholar
Oh, J.-H. & van Pijkeren, J.-P. CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res. 42, e131 (2014).
Google Scholar
Cha, R. S., Zarbl, H., Keohavong, P. & Thilly, W. G. Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. Genome Res. 2, 14–20 (1992).
Google Scholar
Gussak, A., Ferrando, M. L., Schrama, M., Van Baarlen, P. & Wells, J. M. Precision genome engineering in Streptococcus suis based on a broad-host-range vector and CRISPR–Cas9 technology. ACS Synth. Biol. 12, 2546–2560 (2023).
Google Scholar
Zaccaria, E. et al. Control of competence for DNA transformation in Streptococcus suis by genetically transferable pherotypes. PLoS ONE 9, e99394 (2014).
Google Scholar
Zhang, L. et al. Development and application of two inducible expression systems for Streptococcus suis. Microbiol. Spectr. 10, e0036322 (2022).
Google Scholar
Van Kessel, J. C. & Hatfull, G. F. Recombineering in Mycobacterium tuberculosis. Nat. Methods 4, 147–152 (2007).
Google Scholar
Van Kessel, J. C. & Hatfull, G. F. Efficient point mutagenesis in mycobacteria using single‐stranded DNA recombineering: characterization of antimycobacterial drug targets. Mol. Microbiol. 67, 1094–1107 (2008).
Google Scholar
Sörensen, M. et al. Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants. J. Microbiol. Methods 83, 211–216 (2010).
Google Scholar
Knödlseder, N. et al. Delivery of a sebum modulator by an engineered skin microbe in mice. Nat. Biotechnol. 42, 1661–1666 (2024).
Google Scholar
Jore, J. P. M., Van Luijk, N., Luiten, R. G. M., Van Der Werf, M. J. & Pouwels, P. H. Efficient transformation system for Propionibacterium freudenreichii based on a novel vector. Appl. Environ. Microbiol. 67, 499–503 (2001).
Google Scholar
Cheng, L. et al. Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages. BMC Microbiol. 18, 19 (2018).
Google Scholar
Pines, G., Freed, E. F., Winkler, J. D. & Gill, R. T. Bacterial recombineering: genome engineering via phage-based homologous recombination. ACS Synth. Biol. 4, 1176–1185 (2015).
Google Scholar
Farzadfard, F., Gharaei, N., Citorik, R. J. & Lu, T. K. Efficient retroelement-mediated DNA writing in bacteria. Cell Syst. 12, 860–872.e5 (2021).
Google Scholar
Filsinger, G. T. et al. A diverse single-stranded DNA-annealing protein library enables efficient genome editing across bacterial phyla. Proc. Natl Acad. Sci. USA 122, e2414342122 (2025).
Google Scholar
Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P. & Kushner, S. R. New method for generating deletions and gene replacements in Escherichia coli. J. Bacteriol. 171, 4617–4622 (1989).
Google Scholar
Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat. Biotechnol. 31, 233–239 (2013).
Google Scholar
Mougiakos, I. et al. Characterizing a thermostable Cas9 for bacterial genome editing and silencing. Nat. Commun. 8, 1647 (2017).
Google Scholar
Walker, J. E. et al. Development of both type I–B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum. Metab. Eng Commun. 10, e00116 (2020).
Adalsteinsson, B. T. et al. Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant. Sci. Rep. 11, 9586 (2021).
Google Scholar
Stukenberg, D., Hoff, J., Faber, A. & Becker, A. NT-CRISPR, combining natural transformation and CRISPR–Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens. Commun. Biol. 5, 265 (2022).
Google Scholar
Tong, Y. et al. Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST. Proc. Natl Acad. Sci. USA 116, 20366–20375 (2019).
Google Scholar
Xia, P.-F. et al. Reprogramming acetogenic bacteria with CRISPR-targeted base editing via deamination. ACS Synth. Biol. 9, 2162–2171 (2020).
Google Scholar
Volke, D. C., Martino, R. A., Kozaeva, E., Smania, A. M. & Nikel, P. I. Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing. Nat. Commun. 13, 3026 (2022).
Google Scholar
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Google Scholar
Tong, Y., Jørgensen, T. S., Whitford, C. M., Weber, T. & Lee, S. Y. A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing. Nat. Commun. 12, 5206 (2021).
Google Scholar
Zhang, H. et al. BacPE: a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases. Nat. Commun. 15, 825 (2024).
Google Scholar
Bhattarai-Kline, S. et al. Recording gene expression order in DNA by CRISPR addition of retron barcodes. Nature 608, 217–225 (2022).
Google Scholar
Lee, G. & Kim, J. Construction of synthetic protein-binding non-genetic DNA systems in living cells. Nat. Chem. https://doi.org/10.1038/s41557-025-02049-7 (2026).
Katoh, K. & Standley, D. M. MAFFT Multiple Sequence Alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Google Scholar
Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010).
Google Scholar
Saltikov, C. W. & Newman, D. K. Genetic identification of a respiratory arsenate reductase. Proc. Natl Acad. Sci. USA 100, 10983–10988 (2003).
Google Scholar
Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3, e3647 (2008).
Google Scholar
Kok, S. D. et al. Rapid and reliable DNA assembly via ligase cycling reaction. ACS Synth. Biol. 3, 97–106 (2014).
Google Scholar
González-Delgado, A. et al. Genome editing of phylogenetically distinct bacteria using cross-species retron-mediated recombineering. NCBI https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1269658 (2026).
