High-throughput analyses of a reconstituted diversity-generating retroelement identify intrinsic and extrinsic determinants of diversification
Irem Unlu, Marina K. Smiley, Vladimir Potapov, Yoan Renoux-Martin, Zhi-Yi Sun,Hoong Chuin Lim
Abstract
Diversity-Generating Retroelements (DGRs) are specialized genetic systems typically harnessed in nature to evolve new molecular recognition. This mechanism, known as mutagenic retrohoming, relies on an error-prone reverse transcriptase (bRT) that introduces errors at template adenines, followed by the incorporation of the resulting mutagenized complementary DNA (cDNA) into a homologous target gene.
Introduction
Life constantly innovates through genetic trial and error. Organisms explore the vast genetic landscape by accumulating mutations to discover novel traits and adapt to changing environments. This exploration can be sped up through increased mutation rates, but these risks compromise genome integrity. Therefore, strategies enabling organisms to mutate rapidly but safely confer a profound evolutionary advantage, especially when a precise adaptation is urgently needed.
Materials and Methods
Plasmid construction
Plasmids were either constructed by GenScript Biotech or generated in-house using NEBuilder HiFi DNA Assembly (NEB, E2621) or NEB KLD Enzyme Mix (NEB, M0554). The sequences of all plasmids used in this study are listed in S2 Table.
Results
Reconstitution of the BPP-1 DGR in E. coli
We set out to reconstitute the archetypal BPP-1 DGR in E. coli, leveraging the extensive genetic tools available in this bacterium to better understand the mechanism of DGR. We encoded the essential BPP-1 DGR components on two plasmids (Fig 2A): pDGR0 carried the avd-TR-brt operon under an arabinose inducible promoter, while pTarget contained a target gene immediately followed by the essential regulatory elements: GC14, IMH, and stem-loop.
Discussion
We demonstrated for the first time that the entire mutagenic retrohoming pathway, spanning from mutagenic cDNA synthesis to target mutagenesis, can be reconstituted in a heterologous bacterial host. This was achieved by simply transplanting the four core components of the BPP-1 DGR (Fig 2).
Acknowledgments
The authors would like to thank Thomas Evans and Jennifer Ong for championing this project in its early stages, members of the Lim lab for providing critical feedback, Mehmet Berkmen and Emily McNutt for providing KEIO strains for gene deletions, and David Rudner for feedback on the abstract.
Citation: Unlu I, Smiley MK, Potapov V, Renoux-Martin Y, Sun Z-Y, Lim HC (2026) High-throughput analyses of a reconstituted diversity-generating retroelement identify intrinsic and extrinsic determinants of diversification. PLoS Genet 22(2): e1012038. https://doi.org/10.1371/journal.pgen.1012038
Editor: Jan-Willem Veening, University of Lausanne: Universite de Lausanne, SWITZERLAND
Received: August 8, 2025; Accepted: January 23, 2026; Published: February 5, 2026
Copyright: © 2026 Unlu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All sequencing data (raw and processed) are deposited and available from NCBI GEO database under accession numbers: GSE303549, GSE303701, GSE304216, and GSE304215.
Funding: When performing this research and drafting this manuscript, IU, MKS, VP, YR, ZS and HCL were employees of New England Biolabs (NEB), Ipswich, MA. NEB provided funding for this research and paid the salaries for IU, MKS, VP, YR, ZS and HCL but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests. When performing this research and drafting this manuscript, IU, MKS, VP, YR, ZS and HCL were employed by New England Biolabs, a supplier of life sciences reagents, including some used in this study. HCL additionally held equity in Manifold Biotechnologies (unrelated to this work). This employment and equity holding do not, however, alter the authors’ commitment to the highest scientific standards and all PLoS ONE policies on data and material sharing.
