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).

Article  CAS  PubMed  PubMed Central  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).

Article  CAS  PubMed  PubMed Central  Google Scholar 

Wannier, T. M. et al. Recombineering and MAGE. Nat. Rev. Methods Primer 1, 7 (2021).

Article  CAS  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).

Article  CAS  PubMed  PubMed Central  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).

Article  CAS  PubMed  PubMed Central  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).

Article  CAS  PubMed  PubMed Central  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).

Article  CAS  PubMed  PubMed Central  Google Scholar 

Wannier, T. M. et al. Improved bacterial recombineering by parallelized protein discovery. Proc. Natl Acad. Sci. USA 117, 13689–13698 (2020).

Article  CAS  PubMed  PubMed Central  Google Scholar 

Filsinger, G. T. et al. Characterizing the portability of phage-encoded homologous recombination proteins. Nat. Chem. Biol. 17, 394–402 (2021).

Article  CAS  PubMed  PubMed Central  Google Scholar 

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

Article  CAS  PubMed  PubMed Central  Google Scholar 

Ronda, C., Pedersen, L. E., Sommer, M. O. A. & Nielsen, A. T. CRMAGE: CRISPR optimized MAGE recombineering. Sci. Rep. 6, 19452 (2016).

Article  CAS  PubMed  PubMed Central  Google Scholar 

Corts, A., Thomason, L. C., Costantino, N. & Court, D. L. Recombineering in non‐model bacteria. Curr. Protoc. 2, e605 (2022).

Article  CAS  PubMed  PubMed Central  Google Scholar 

Isaacs, F. J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348–353 (2011).

Article  CAS  PubMed  PubMed Central  Google Scholar 

Millman, A. et al. Bacterial retrons function in anti-phage defense. Cell 183, 1551–1561.e12 (2020).

Article  CAS  PubMed  Google Scholar 

Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084 (2020).

Article  CAS  PubMed  PubMed Central  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).

Article  CAS  PubMed  PubMed Central  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).

Article  CAS  PubMed  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).

Article  CAS  PubMed  PubMed Central  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).

Article  CAS  PubMed  PubMed Central  Google Scholar 

Carabias, A. et al. Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages. Mol. Cell 84, 2185–2202.e12 (2024).

Article  CAS  PubMed  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).

Article  PubMed  PubMed Central  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).

Article  CAS  PubMed  PubMed Central  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).

Article  CAS  PubMed  Google Scholar 

Fishman, C. B. et al. Continuous multiplexed phage genome editing using recombitrons. Nat. Biotechnol. 43, 1299–1310 (2025).

Article  CAS  PubMed  Google Scholar 

Liu, W. et al. Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli. Nucleic Acids Res. 51, 8293–8307 (2023).

Article  CAS  PubMed  PubMed Central  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).

Article  PubMed  PubMed Central  Google Scholar 

Simon, A. J., Morrow, B. R. & Ellington, A. D. Retroelement-based genome editing and evolution. ACS Synth. Biol. 7, 2600–2611 (2018).

Article  CAS  PubMed  Google Scholar 

Liu, W. et al. Intracellularly synthesized ssDNA for continuous genome engineering. Trends Biotechnol. 43, 1356–1370 (2025).

Article  CAS  PubMed  Google Scholar 

Ni, Y. et al. Reducing competition between msd and genomic DNA improves retron editing efficiency. EMBO Rep. 25, 5316–5330 (2024).

Article  PubMed  PubMed Central  Google Scholar 

Khan, A. G. et al. An experimental census of retrons for DNA production and genome editing. Nat. B

Comments (0)

No login
gif