Abstract
Retrons are bacterial immune systems that use reverse-transcribed DNA (RT-DNA) to detect phage infection. They are also deployed for genome editing, where they are modified so that the RT-DNA encodes an editing donor. Retrons are common in bacterial genomes, and thousands of unique retrons have been predicted bioinformatically. However, few have been characterized experimentally. We add to the corpus of experimentally studied retrons, finding 62 empirically determined, natural RT-DNAs that are not predictable from the retron sequence alone. We synthesize >100 previously untested retrons to identify the natural sequence of RT-DNA they produce, quantify their RT-DNA production and test the relative efficacy of editing using retron-derived donors to edit bacterial, phage and human genomes. We observe large diversity in RT-DNA production and editing rates across retrons, finding that top-performing editors are drawn from a subset of the retron phylogeny and outperform those used in previous studies, reaching precise editing rates of up to 40% in human cells.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
All data supporting the findings of this study are available within the article and its Supplementary Information or will be made available from the authors upon request. Sequencing data associated with this study are available on National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) (PRJNA1047666)34.
Code availability
Custom code to process or analyze data from this study is available via GitHub (https://github.com/Shipman-Lab/Retron-Census)35.
Change history
30 September 2024
In the version of this article initially published, Supplementary Data 1 was missing, while the three references to Supplementary Data 1 now in the text originally referred to Supplementary Table 1, twice, and Supplementary Table 6. The changes are made in the HTML and PDF versions of the article.
References
Yee, T., Furuichi, T., Inouye, S. & Inouye, M. Multicopy single-stranded DNA isolated from a gram-negative bacterium, Myxococcus xanthus. Cell 38, 203–209 (1984).
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).
Hsu, M. Y., Eagle, S. G., Inouye, M. & Inouye, S. Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli. J. Biol. Chem. 267, 13823–13829 (1992).
Shimamoto, T., Inouye, M. & Inouye, S. The formation of the 2′,5′-phosphodiester linkage in the cDNA priming reaction by bacterial reverse transcriptase in a cell-free system. J. Biol. Chem. 270, 581–588 (1995).
Shimamoto, T., Kawanishi, H., Tsuchiya, T., Inouye, S. & Inouye, M. In vitro synthesis of multicopy single-stranded DNA, using separate primer and template RNAs, by Escherichia coli reverse transcriptase. J. Bacteriol. 180, 2999–3002 (1998).
Simon, A. J., Ellington, A. D. & Finkelstein, I. J. Retrons and their applications in genome engineering. Nucleic Acids Res. 47, 11007–11019 (2019).
Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084 (2020).
Millman, A. et al. Bacterial retrons function in anti-phage defense. Cell 183, 1551–1561 (2020).
Bobonis, J. et al. Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems. Nature 609, 144–150 (2022).
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).
Wang, Y. et al. Cryo-EM structures of Escherichia coli Ec86 retron complexes reveal architecture and defence mechanism. Nat. Microbiol. 7, 1480–1489 (2022).
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).
Farzadfard, F. & Lu, T. K. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations. Science 346, 1256272 (2014).
Sharon, E. et al. Functional genetic variants revealed by massively parallel precise genome editing. Cell 175, 544–557 (2018).
Bhattarai-Kline, S. et al. Recording gene expression order in DNA by CRISPR addition of retron barcodes. Nature 608, 217–225 (2022).
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).
Lee, G. & Kim, J. Engineered retrons generate genome-independent protein-binding DNA for cellular control. Preprint at bioRxiv https://doi.org/10.1101/2023.09.27.556556 (2023).
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).
Kong, X. et al. Precise genome editing without exogenous donor DNA via retron editing system in human cells. Protein Cell 12, 899–902 (2021).
Zhao, B., Chen, S. A., Lee, J. & Fraser, H. B. Bacterial retrons enable precise gene editing in human cells. CRISPR J. 5, 31–39 (2022).
Liu, W. et al. Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli. Nucleic Acids Res. 51, 8293–8307 (2023).
Fishman, C. B. et al. Continuous multiplexed phage genome editing using recombitrons. Nat. Biotechnol. https://doi.org/10.1038/s41587-024-02370-5 (2024).
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. https://doi.org/10.1038/s41589-024-01665-7 (2024).
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).
Wimberger, S. et al. Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing. Nat. Commun. 14, 4761 (2023).
Lampson, B. C., Inouye, M. & Inouye, S. Retrons, msDNA, and the bacterial genome. Cytogenet. Genome Res. 110, 491–499 (2005).
Kim, K., Jeong, D. & Lim, D. A mutational study of the site-specific cleavage of EC83, a multicopy single-stranded DNA (msDNA): nucleotides at the msDNA stem are important for its cleavage. J. Bacteriol. 179, 6518–6521 (1997).
Lease, R. A. & Yee, T. Early events in the synthesis of the multicopy single-stranded DNA–RNA branched copolymer of Myxococcus xanthus. J. Biol. Chem. 266, 14497–14503 (1991).
Azam, A. H. et al. Viruses encode tRNA and anti-retron to evade bacterial immunity. Preprint at bioRxiv https://doi.org/10.1101/2023.03.15.532788 (2023).
Wannier, T. M. et al. Improved bacterial recombineering by parallelized protein discovery. Proc. Natl Acad. Sci. USA 117, 13689–13698 (2020).
Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).
Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L. & Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR–Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).
Kropinski, A. M., Mazzocco, A., Waddell, T. E., Lingohr, E. & Johnson, R. P. Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol. Biol. 501, 69–76 (2009).
Khan, A. G. et al. An experimental census of retrons for DNA production and genome editing. www.ncbi.nlm.nih.gov/bioproject/PRJNA1047666/ (2024).
Fang, R. & Shipman, S. Shipman-Lab/Retron-Census. GitHub github.com/Shipman-Lab/Retron-Census (2024).
Acknowledgements
This work was supported by funding from the National Science Foundation (MCB 2137692), the National Institute of Biomedical Imaging and Bioengineering (R21EB031393) and the National Institute of General Medical Sciences (1DP2GM140917), with research support from Retronix Bio. S.L.S. is a Chan Zuckerberg Biohub San Francisco Investigator and acknowledges additional funding support from the L.K. Whittier Foundation and the Pew Biomedical Scholars Program. A.G.-D. is supported by the California Institute of Regenerative Medicine (CIRM) scholar program. S.C.L. is supported by a Berkeley Fellowship for Graduate Study. R.F.F. is supported by a UCSF Discovery Fellowship. K.D.C. is supported by a National Science Foundation Graduate Research Fellowship and a UCSF Discovery Fellowship. We would like to thank A. Pico and the Gladstone Bioinformatics Core for assistance with data database management, as well as K. Zhang and D. Wen for comments on the manuscript.
Author information
Authors and Affiliations
Contributions
S.C.L., A.G.-D. and S.L.S. conceived the study. M.R.-M. and A.G.-D. performed the experiments in bacteria and phages. A.G.K. performed experiments on human cells. K.D.C. performed the STING assay. S.L.S., A.G.K., M.R.-M., A.G.-D., S.C.L., R.F.F. and K.D.C. analyzed the data. A.G.K., M.R.-M., A.G.-D. and S.L.S. wrote the manuscript with inputs from all authors.
Corresponding author
Ethics declarations
Competing interests
S.L.S. is a cofounder of Retronix Bio and Sprint Synthesis. A.G.-D., S.C.L. and S.L.S. are named inventors on patent applications related to the technologies described in this work that are assigned to the Gladstone Institutes and the University of California, San Francisco. The remaining authors declare no competing interests.
Peer review
Peer review information
Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 RT-DNA production by subtype.
Related to Fig. 1. RT-DNA production relative to Eco1 by clade, separated by subtype. Bars show mean ± SEM, and circles are individual retrons.
Extended Data Fig. 2 Motif analysis around branching guanosine.
Related to Fig. 2. Probability logo of ncRNA nucleotides adjacent to the branching guanosine for retrons that produce RT-DNA.
Extended Data Fig. 3 RT-DNA production versus phage recombineering.
Related to Fig. 3. RT-DNA production versus phage lambda recombineering rates (Pearson correlation (r2 = 0.001926, P = 0.8766, two-sided)).
Extended Data Fig. 4 Validation of human editing experimental design conditions.
Related to Fig. 4. a, Demultiplexed editing percentage by retron-Eco1 (Mestre-1550) comparing the use of an H1 and U6 promoter to drive ncRNA/sgRNA (unpaired, two-sided T-test, P = 0.6256). Bars are mean ± SEM, and closed circles are biological replicates (N ≥ 3). b, Fold change in STING response after 48 hours of retron induction or 2′–3′ cGAMP (0.003–30 μg/mL) relative to a mock-transfection control with 0 μg/mL 2′–3′ cGAMP. Open circles are biological replicates. Each biological replicate is the mean of three technical replicates. c, Comparison of demultiplexed editing rates of editrons in the constitutive and inducible Cas9 cell lines (Pearson correlation, r2 = 0.4196, P < 0.0001, two-sided). d, Average demultiplexed editing rates across retron subtypes. Bars are mean ± SEM, and circles are individual retrons.
Extended Data Fig. 5 Additional quantification of human editrons in follow-up testing.
Related to Fig. 5. a, Editron architecture with fused ncRNA/gRNA. b, Precise editing for 11 retrons, including retron-Eco1 in the fused architecture (1550, in blue). Bars are mean ± SEM, and closed circles are each of three biological replicates. c, Indel percentage for eleven retrons, including retron-Eco1 in the fused architecture (1550, in blue). Bars are mean ± SEM, and closed circles are each of three biological replicates. d, Indel versus precise editing percentage for the fused architecture, and mean ± SEM for three biological replicates. e, Editron architecture with split ncRNA and gRNA. f, Precise editing for 11 retrons, including retron-Eco1 in the split architecture (1550, in blue). Bars are mean ± SEM, and closed circles are each of three biological replicates. Note that these data are replotted in Fig. 5b. g, Indel percentage for eleven retrons, including retron-Eco1 in the split architecture (1550, in blue). Bars are mean ± SEM, and closed circles are each of three biological replicates. h, Indel versus precise editing percentage for the split architecture, and mean ± SEM for three biological replicates. i, Substitutions per base on edited reads beyond the errors on wild-type reads. Open circles are three biological replicates, each compared to wild-type reads from the same sample. j, Deletions per base on edited reads beyond the errors on wild-type reads. Open circles are three biological replicates, each compared to wild-type reads from the same sample. In cases where there are fewer than three points, there were not enough errors to quantify the frequency. k, Insertions per base on edited reads beyond the errors on wild-type reads. Open circles are three biological replicates, each compared to wild-type reads from the same sample. In cases where there are fewer than three points, there were not enough errors to quantify the frequency.
Extended Data Fig. 6 Bacterial RT-DNA production versus human editing.
Additional data related to Fig. 4. Bacterial RT-DNA production compared with human demultiplexed editing.
Supplementary information
Supplementary Information
Supplementary Fig. 1 and Supplementary Tables 2–5.
Supplementary Table 1
Retrons.
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.
About this article
Cite this article
Khan, A.G., Rojas-Montero, M., González-Delgado, A. et al. An experimental census of retrons for DNA production and genome editing. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02384-z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41587-024-02384-z
