Diversity-generating retroelements for programmable targeted hypermutagenesis

Packer, M. S. & Liu, D. R. Methods for the directed evolution of proteins. Nat. Rev. Genet. 16, 379–394 (2015).
Google Scholar
Lynch, M. Evolution of the mutation rate. Trends Genet. 26, 345–352 (2010).
Google Scholar
Morrison, M. S., Podracky, C. J. & Liu, D. R. The developing toolkit of continuous directed evolution. Nat. Chem. Biol. 16, 610–619 (2020).
Google Scholar
Molina, R. S. et al. In vivo hypermutation and continuous evolution. Nat. Rev. Methods Primers 2, 36 (2022).
Google Scholar
Ravikumar, A., Arzumanyan, G. A., Obadi, M. K. A., Javanpour, A. A. & Liu, C. C. Scalable, continuous evolution of genes at mutation rates above genomic error thresholds. Cell 175, 1946–1957 (2018).
Google Scholar
Tian, R. et al. Engineered bacterial orthogonal DNA replication system for continuous evolution. Nat. Chem. Biol. 19, 1504–1512 (2023).
Google Scholar
Tian, R. et al. Establishing a synthetic orthogonal replication system enables accelerated evolution in E. coli. Science 383, 421–426 (2024).
Google Scholar
Halperin, S. O. et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature 560, 248–252 (2018).
Google Scholar
Hurtado, J. E. et al. Nickase fidelity drives EvolvR-mediated diversification in mammalian cells. Nat. Commun. 16, 3723 (2025).
Google Scholar
Moore, C. L., Papa, L. J. III & Shoulders, M. D. A processive protein chimera introduces mutations across defined DNA regions in vivo. J. Am. Chem. Soc. 140, 11560–11564 (2018).
Google Scholar
Janeway, C. A., Jr, Travers, P., Walport, M. & Shlomchik, M. J. The Generation of Diversity in Immunoglobulins 5th edn (Garland Science, 2001).
Liu, M. et al. Reverse transcriptase-mediated tropism switching in Bordetella bacteriophage. Science 295, 2091–2094 (2002).
Google Scholar
Doulatov, S. et al. Tropism switching in Bordetella bacteriophage defines a family of diversity-generating retroelements. Nature 431, 476–481 (2004).
Google Scholar
Paul, B. G. et al. Retroelement-guided protein diversification abounds in vast lineages of Bacteria and Archaea. Nat. Microbiol. 2, 1–7 (2017).
Google Scholar
Roux, S. et al. Ecology and molecular targets of hypermutation in the global microbiome. Nat. Commun. 12, 3076 (2021).
Google Scholar
Macadangdang, B. R. et al. Targeted protein evolution in the gut microbiome by diversity-generating retroelements. Science 390, eadv2111 (2025).
Google Scholar
Wu, L. et al. Diversity-generating retroelements: natural variation, classification and evolution inferred from a large-scale genomic survey. Nucleic Acids Res. 46, 11–24 (2018).
Google Scholar
Dai, W. et al. Three-dimensional structure of tropism-switching Bordetella bacteriophage. Proc. Natl Acad. Sci. USA 107, 4347–4352 (2010).
Google Scholar
Guo, H. et al. Target site recognition by a diversity-generating retroelement. PLoS Genet. 7, e1002414 (2011).
Google Scholar
Handa, S. et al. Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex. Nucleic Acids Res. 46, 9711–9725 (2018).
Google Scholar
McMahon, S. A. et al. The C-type lectin fold as an evolutionary solution for massive sequence variation. Nat. Struct. Mol. Biol. 12, 886–892 (2005).
Google Scholar
Handa, S. et al. RNA control of reverse transcription in a diversity-generating retroelement. Nature 638, 1122–1129 (2025).
Google Scholar
Macadangdang, B. R., Makanani, S. K. & Miller, J. F. Accelerated Evolution by Diversity-Generating Retroelements. Annu Rev Microbiol. 76, 389–411 (2022).
Google Scholar
Handa, S., Paul, B. G., Miller, J. F., Valentine, D. L. & Ghosh, P. Conservation of the C-type lectin fold for accommodating massive sequence variation in archaeal diversity-generating retroelements. BMC Struct. Biol. 16, 13 (2016).
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
Simon, A. J., Morrow, B. R. & Ellington, A. D. Retroelement-based genome editing and evolution. ACS Synth. Biol. 7, 2600–2611 (2018).
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
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
Fishman, C. B. et al. Continuous multiplexed phage genome editing using recombitrons. Nat Biotechnol. 43, 1299–1310 (2025).
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
Wannier, T. M. et al. Improved bacterial recombineering by parallelized protein discovery. Proc. Natl Acad. Sci. USA 117, 13689–13698 (2020).
Google Scholar
Naorem, S. S. et al. DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA. Proc. Natl Acad. Sci. USA 114, E10187–E10195 (2017).
Google Scholar
Kou, R. et al. Benefits and challenges with applying unique molecular identifiers in next generation sequencing to detect low frequency mutations. PLoS One 11, e0146638 (2016).
Google Scholar
Handa, S., Reyna, A., Wiryaman, T. & Ghosh, P. Determinants of adenine-mutagenesis in diversity-generating retroelements. Nucleic Acids Res. 49, 1033–1045 (2021).
Google Scholar
Wannier, T. M. et al. Recombineering and MAGE. Nat. Rev. Methods Primers 1, 7 (2021).
Google Scholar
Fishman, C. B. et al. Continuous multiplexed phage genome editing using recombitrons. Nat. Biotechnol. 43, 1299–1310 (2025).
Google Scholar
Lorenz, R. et al. ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26 (2011).
Google Scholar
Brockhurst, M. A., Koskella, B. & Zhang, Q. G. Bacteria–phage antagonistic coevolution and the implications for phage therapy. In Bacteriophages (eds Harper, D. R., Abedon, S. T., Burrowes, B. H. & McConville, M.L.) (Springer, 2017).
Borin, J. M. et al. Rapid bacteria–phage coevolution drives the emergence of multiscale networks. Science 382, 674–678 (2023).
Google Scholar
Meyer, J. R. et al. Repeatability and contingency in the evolution of a key innovation in phage lambda. Science 335, 428–432 (2012).
Google Scholar
Andrews, B. & Fields, S. Balance between promiscuity and specificity in phage λ host range. ISME J. 15, 2195–2205 (2021).
Google Scholar
Ge, X. & Wang, J. Structural mechanism of bacteriophage lambda tail’s interaction with the bacterial receptor. Nat. Commun. 15, 4185 (2024).
Google Scholar
Salema, V. et al. Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two β-domains of opposite topologies. PLoS ONE 8, e75126 (2013).
Google Scholar
Salema, V. & Fernández, L. Á Escherichia coli surface display for the selection of nanobodies. Microb. Biotechnol. 10, 1468–1484 (2017).
Google Scholar
Casasnovas, J. M. et al. Nanobodies protecting from lethal SARS-CoV-2 infection target receptor binding epitopes preserved in virus variants other than Omicron. Front. Immunol. 13, 863831 (2022).
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
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
Ni, Y. et al. Reducing competition between msd and genomic DNA improves retron editing efficiency. EMBO Rep. 25, 5316–5330 (2024).
Google Scholar
Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (2011).
Google Scholar
Miller, S. M., Wang, T. & Liu, D. R. Phage-assisted continuous and non-continuous evolution. Nat. Protoc. 15, 4101–4127 (2020).
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
St-Pierre, F. et al. One-step cloning and chromosomal integration of DNA. ACS Synth. Biol. 2, 537–541 (2013).
Google Scholar
Thomason, L. C., Costantino, N. & Court, D. L. E. coli genome manipulation by P1 transduction. Curr. Protoc. Mol. Biol. Chapter 1, 1.17.1–1.17.8 (2007).
Google Scholar
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
Google Scholar
Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden Gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4, e5553 (2009).
Google Scholar
Hartley, J. L., Temple, G. F. & Brasch, M. A. DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795 (2000).
Google Scholar
Jakočiūnė, D. & Moodley, A. A rapid bacteriophage DNA extraction method. Methods Protoc. 1, 27 (2018).
Google Scholar
Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2022).
Google Scholar
Clement, K., Farouni, R., Bauer, D. E. & Pinello, L. AmpUMI: design and analysis of unique molecular identifiers for deep amplicon sequencing. Bioinformatics 34, i202–i210 (2018).
Google Scholar
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
Google Scholar




