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Continuous Multiplexed Phage Genome Editing Using Recombitrons

Fishman, Chloe B. and Crawford, Kate D. and Bhattarai-Kline, Santi and Zhang, Karen and Delgado-Gonzalez, Alejandro and Shipman, Seth L. (2023) Continuous Multiplexed Phage Genome Editing Using Recombitrons. . (Unpublished)

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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.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Fishman, Chloe B.0000-0002-4698-4469
Bhattarai-Kline, Santi0000-0003-4896-7109
Zhang, Karen0000-0003-1177-635X
Delgado-Gonzalez, Alejandro0000-0002-8357-3204
Shipman, Seth L.0000-0003-3130-8043
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.
Funding AgencyGrant Number
Gary and Eileen Morgenthaler FundUNSPECIFIED
L. K. Whittier FoundationUNSPECIFIED
Pew Charitable TrustUNSPECIFIED
Record Number:CaltechAUTHORS:20230327-442827000.4
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:120414
Deposited By: George Porter
Deposited On:30 Mar 2023 03:24
Last Modified:30 Mar 2023 03:24

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