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Published March 30, 2023 | Submitted + Supplemental Material
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Continuous Multiplexed Phage Genome Editing Using Recombitrons

Abstract

Bacteriophages, which naturally shape bacterial communities, can be co-opted as a biological technology to help eliminate pathogenic bacteria from our bodies and food supply. Phage genome editing is a critical tool to engineer more effective phage technologies. However, editing phage genomes has traditionally been a low efficiency process that requires laborious screening, counter selection, or in vitro construction of modified genomes. These requirements impose limitations on the type and throughput of phage modifications, which in turn limit our knowledge and potential for innovation. Here, we present a scalable approach for engineering phage genomes using recombitrons: modified bacterial retrons that generate recombineering donor DNA paired with single stranded binding and annealing proteins to integrate those donors into phage genomes. This system can efficiently create genome modifications in multiple phages without the need for counterselection. Moreover, the process is continuous, with edits accumulating in the phage genome the longer the phage is cultured with the host, and multiplexable, with different editing hosts contributing distinct mutations along the genome of a phage in a mixed culture. In lambda phage, as an example, recombitrons yield single-base substitutions at up to 99% efficiency and up to 5 distinct mutations installed on a single phage genome, all without counterselection and only a few hours of hands-on time.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. Work was supported by funding from the National Science Foundation (MCB 2137692), the National Institute of Biomedical Imaging and Bioengineering (R21EB031393), the Gary and Eileen Morgenthaler Fund, and the National Institute of General Medical Sciences (1DP2GM140917). S.L.S. acknowledges additional funding support from the L.K. Whittier Foundation and the Pew Biomedical Scholars Program. Author Contributions. C.B.F., S.B.-K., and S.L.S. conceived the study and, with K.D.C., K.A.Z., and A.G.-D., outlined the scope of the project and designed experiments. C.B.F. developed phage handling and editing protocols. Experiments were performed and analyzed by C.B.F. (Fig1 e-h, Fig2, Fig4, Supp Fig1 b-h, Supp Fig2, Supp Fig4), K.D.C. (Fig3, Supp Fig3), S.B.-K. (Fig1 b-d, Supp Fig1 a), and K.A.Z (Fig3, Supp Fig3). C.B.F. and S.L.S. wrote the manuscript, with input from all authors. 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 in the NCBI SRA (PRJNA933262). Code Availability. Custom code to process or analyze data from this study will be made available on GitHub prior to peer-reviewed publication. Competing Interest Statement. 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.

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Submitted - 2023.03.24.534024v1.full.pdf

Supplemental Material - media-1.xlsx

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Additional details

Created:
August 20, 2023
Modified:
October 18, 2023