.2024 Sep 5:10.1038/s41587-024-02370-5.

doi: 10.1038/s41587-024-02370-5. Online ahead of print.

Continuous multiplexed phage genome editing using recombitrons

Chloe B Fishman^#¹, Kate D Crawford^#^{1 2}, Santi Bhattarai-Kline^#^{1 3}, Darshini Poola^#^{1 4}, Karen Zhang^{1 2}, Alejandro González-Delgado¹, Matías Rojas-Montero¹, Seth L Shipman^{5 6 7}

Affiliations

¹ Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.
² Graduate Program in Bioengineering, University of California, San Francisco and Berkeley, CA, USA.
³ UCLA-Caltech Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.
⁴ Indian Institute of Science Education and Research (IISER), Pune, India.
⁵ Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA. seth.shipman@gladstone.ucsf.edu.
⁶ Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA. seth.shipman@gladstone.ucsf.edu.
⁷ Chan Zuckerberg Biohub, San Francisco, CA, USA. seth.shipman@gladstone.ucsf.edu.

^# Contributed equally.

PMID:39237706
PMCID:PMC11880354 (available on2026-03-05)
DOI: 10.1038/s41587-024-02370-5

Continuous multiplexed phage genome editing using recombitrons

Chloe B Fishman et al. Nat Biotechnol.2024.

.2024 Sep 5:10.1038/s41587-024-02370-5.

doi: 10.1038/s41587-024-02370-5. Online ahead of print.

Authors

Chloe B Fishman^#¹, Kate D Crawford^#^{1 2}, Santi Bhattarai-Kline^#^{1 3}, Darshini Poola^#^{1 4}, Karen Zhang^{1 2}, Alejandro González-Delgado¹, Matías Rojas-Montero¹, Seth L Shipman^{5 6 7}

Affiliations

¹ Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.
² Graduate Program in Bioengineering, University of California, San Francisco and Berkeley, CA, USA.
³ UCLA-Caltech Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.
⁴ Indian Institute of Science Education and Research (IISER), Pune, India.
⁵ Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA. seth.shipman@gladstone.ucsf.edu.
⁶ Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA. seth.shipman@gladstone.ucsf.edu.
⁷ Chan Zuckerberg Biohub, San Francisco, CA, USA. seth.shipman@gladstone.ucsf.edu.

^# Contributed equally.

PMID:39237706
PMCID:PMC11880354 (available on2026-03-05)
DOI: 10.1038/s41587-024-02370-5

Abstract

Bacteriophage genome editing can enhance the efficacy of phages to eliminate pathogenic bacteria in patients and in the environment. However, current methods for editing phage genomes require laborious screening, counterselection or in vitro construction of modified genomes. Here, we present a scalable approach that uses modified bacterial retrons called recombitrons to generate recombineering donor DNA paired with single-stranded binding and annealing proteins for integration into phage genomes. This system can efficiently create genome modifications in multiple phages without the need for counterselection. The approach also supports larger insertions and deletions, which can be combined with simultaneous counterselection for >99% efficiency. Moreover, we show that the process is continuous, with more edits accumulating the longer the phage is cultured with the host, and multiplexable. We install up to five distinct mutations on a single lambda phage genome without counterselection in only a few hours of hands-on time and identify a residue-level epistatic interaction in the T7 gp17 tail fiber.

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Conflict of interest statement

Competing Interests

C.B.F., S.B.K., and S.L.S. are named inventors on a patent application related to the technologies described in this work.

Update of

Continuous Multiplexed Phage Genome Editing Using Recombitrons.
Fishman CB, Crawford KD, Bhattarai-Kline S, Zhang K, González-Delgado A, Shipman SL.Fishman CB, et al.bioRxiv [Preprint]. 2023 Mar 25:2023.03.24.534024. doi: 10.1101/2023.03.24.534024.bioRxiv. 2023.Update in:Nat Biotechnol. 2024 Sep 5. doi: 10.1038/s41587-024-02370-5.PMID:36993281Free PMC article.Updated.Preprint.

Cited by

An experimental census of retrons for DNA production and genome editing.
Khan AG, Rojas-Montero M, González-Delgado A, Lopez SC, Fang RF, Shipman SL.Khan AG, et al.bioRxiv [Preprint]. 2024 Jan 26:2024.01.25.577267. doi: 10.1101/2024.01.25.577267.bioRxiv. 2024.Update in:Nat Biotechnol. 2024 Sep 17. doi: 10.1038/s41587-024-02384-z.PMID:38328236Free PMC article.Updated.Preprint.
An efficient, scarless, selection-free technology for phage engineering.
Goren MG, Mahata T, Qimron U.Goren MG, et al.RNA Biol. 2023 Jan;20(1):830-835. doi: 10.1080/15476286.2023.2270344. Epub 2023 Oct 16.RNA Biol. 2023.PMID:37846029Free PMC article.
Simultaneous multi-site editing of individual genomes using retron arrays.
González-Delgado A, Lopez SC, Rojas-Montero M, Fishman CB, Shipman SL.González-Delgado A, et al.Nat Chem Biol. 2024 Nov;20(11):1482-1492. doi: 10.1038/s41589-024-01665-7. Epub 2024 Jul 9.Nat Chem Biol. 2024.PMID:38982310
Simultaneous multi-site editing of individual genomes using retron arrays.
González-Delgado A, Lopez SC, Rojas-Montero M, Fishman CB, Shipman SL.González-Delgado A, et al.bioRxiv [Preprint]. 2023 Jul 17:2023.07.17.549397. doi: 10.1101/2023.07.17.549397.bioRxiv. 2023.Update in:Nat Chem Biol. 2024 Nov;20(11):1482-1492. doi: 10.1038/s41589-024-01665-7.PMID:37503029Free PMC article.Updated.Preprint.
High Throughput Variant Libraries and Machine Learning Yield Design Rules for Retron Gene Editors.
Crawford KD, Khan AG, Lopez SC, Goodarzi H, Shipman SL.Crawford KD, et al.bioRxiv [Preprint]. 2024 Jul 9:2024.07.08.602561. doi: 10.1101/2024.07.08.602561.bioRxiv. 2024.Update in:Nucleic Acids Res. 2025 Jan 11;53(2):gkae1199. doi: 10.1093/nar/gkae1199.PMID:39026735Free PMC article.Updated.Preprint.

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Continuous multiplexed phage genome editing using recombitrons

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Continuous multiplexed phage genome editing using recombitrons

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