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Continuous multiplexed phage genome editing using recombitrons

Nature Biotechnology (2024)Cite this article

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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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Fig. 1: Recombitrons target phage genomes for continuous editing.
Fig. 2: Optimizing recombitron parameters.
Fig. 3: Insertions and deletions using recombitrons.
Fig. 4: Multiplexed phage engineering using recombitrons.
Fig. 5: Combinatorial mutations of the T7 tail fiber.

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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 from the NCBI SRA (PRJNA933262).

Code availability

Custom code to process or analyze data from this study is available from GitHub (https://github.com/Shipman-Lab/Multiplexed_Phage_Recombitrons).

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Acknowledgements

This 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. 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. K.D.C. and K.Z. were supported by National Science Foundation Graduate Research Fellowships and University of California, San Francisco Discovery Fellowships. A.G.-D. was supported by the California Institute of Regenerative Medicine scholar program.

Author information

Author notes

  1. These authors contributed equally: Chloe B. Fishman, Kate D. Crawford, Santi Bhattarai-Kline, Darshini Poola.

Authors and Affiliations

  1. Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA

    Chloe B. Fishman, Kate D. Crawford, Santi Bhattarai-Kline, Darshini Poola, Karen Zhang, Alejandro González-Delgado, Matías Rojas-Montero & Seth L. Shipman

  2. Graduate Program in Bioengineering, University of California, San Francisco and Berkeley, CA, USA

    Kate D. Crawford & Karen Zhang

  3. UCLA-Caltech Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, CA, USA

    Santi Bhattarai-Kline

  4. Indian Institute of Science Education and Research (IISER), Pune, India

    Darshini Poola

  5. Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA

    Seth L. Shipman

  6. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Seth L. Shipman

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Contributions

C.B.F., S.B.-K. and S.L.S. conceptualized 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 the phage handling and editing protocols. Experiments were performed and analyzed by C.B.F. (Figs. 1e,f,i, 2a–k and 4 and Extended Data Figs. 1c–m, 2a and 4), K.D.C. (Figs. 2l,m, 3 and 5 and Extended Data Figs. 1b and 3), S.B.-K. (Fig. 1b–d and Extended Data Fig. 1a), D.P. (Fig. 5), K.A.Z. (Figs. 1g and 3 and Extended Data Figs. 1n and 3) and A.G.-D. (Fig. 2n and Extended Data Fig. 2b–d). C.B.F. and S.L.S. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Seth L. Shipman.

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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. S.L.S. is a cofounder of Retronix Bio and Sprint Synthesis. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Accompaniment to Fig. 1.

a. Left: Edited phage T2 genomes (%). With forward (blue) or reverse (purple) RT-DNA. Open circles are three biological replicates, closed circles are means. Right: Recombitron editing at site 143,279 (R) (±SD) versus a dRT control (unpaired, two-sided t-test, P = 0.77). b. Editing of wild-type and mutant T4 without modified cytosines, shown as in a (N = 3) (two-way ANOVA, effect of modified bases, P = 0.0061). c. Editing (%) of lambda and lambda_ΔcI in different host strains at site 14,070 (R). Open circles show 3 (wt in bSLS.114), 5 (ΔcI in bSLS.114), and 2 (ΔcI in bCF.5) biological replicates, closed circles show the mean (one-way ANOVA, effect of strain/phage, P = 0.2421). d. Editing (%) of lambda without induction of CspRecT at site 14,126, shown as in a (N = 3). e. Titer (PFU/mL) of phage lambda, T7, and T5 after propagation through host cells of different conditions, compared to amount of phage added to the culture, without recombitrons (open black circles), with uninduced recombitrons (open pink circles), with induced recombitrons (closed pink circles), and with induced recombitrons targeting a different phage (closed blue circles). Individual biological replicates are shown. f. Editing (%) of lambda, T7, and T5 from the induced, on-target recombitron condition in panel d, shown as in a (N = 3). g. Editing (%) of T7 with supplemental expression of lambda genes gam or beta, shown as in a (N = 3, one-way ANOVA P < 0.0001). h. Editing (%) of lamda with supplemental expression of lambda genes gam or beta, shown as in a (N = 3, one-way ANOVA P = 0.0904). i. Editing (%) of T7 with supplemental expression of E. coli SSB and lambda genes gam or beta, shown as in a (N = 3, one-way ANOVA P < 0.0001). j. Editing (%) of lambda with supplemental expression of E. coli SSB and lambda genes gam or beta, as in a (N = 3, one-way ANOVA P < 0.0001). k. Editing (%) of lambda at site 14126 (F) compared to editing with supplemental expression of T5 SSB, shown as in a (N = 3). l. Editing (%) of T7 at site 22872 (R) compared to editing with supplemental expression of T5 SSB, shown as in a (N = 3). m. Editing (%) of T5 at site 88634 (F) with supplemental expression of E. coli SSB, T7 SSB, or T5 SSB, shown as in a (N = 3). n. Editing (%) of phages from the basal collection, Bas46 (A19798T) and Bas47 (A6332G), that contain modified bases with the RT induced (+) or uninduced (-), as in f (N = 3).

Extended Data Fig. 2 Accompaniment to Fig. 2.

a. Rate of acquiring only the scanning edit in lambda when donors contain both scanning and central edits. (open circles are biological replicates, closed circles are the mean). b. PAGE analysis of retron RT-DNA in different E. coli strains. c. Editing (%) of lambda with editing cultures started at different initial multiplicities of infection (MOI), using MG1655 (Δexo1,ΔrecJ) as the editing host (one-way ANOVA P < 0.0001) (open circles are 3 biological replicates, closed circles are the mean). d. Editing (%) of lambda with editing cultures incubated at different temperatures, using MG1655 (Δexo1,ΔrecJ) as the editing host (unpaired, two-sided t-test P = 0.0013) (open circles are 3 biological replicates, closed circles are the mean).

Extended Data Fig. 3 Accompaniment to Fig. 3.

a. Comparison of edited phages measure by amplicon (Illumina) or amplification-free (Oxford Nanopore) sequencing. Open orange circles represent biological replicates of amplicon data and filled orange circle represents the mean. Filled blue circle represents the aggregate nanopore data from three replicates. b. Coverage of the editing site in long-read nanopore sequencing for deletions in which we observe editing. c. Coverage of the editing site in long-read nanopore sequencing for deletions in which we do not observe any edits. Estimated limit of detection for these samples is calculated by dividing 100 by the coverage of the site. d. Examples of nanopore reads for different deletion conditions. e. Coverage of the editing site in long-read nanopore sequencing for large insertions, for which we do not observe any edits. Estimated limit of detection for these samples is calculated by dividing 100 by the coverage of the site.

Extended Data Fig. 4 Accompaniment to Fig. 4.

Editing (%) from Sanger sequencing of plaques at each site from mixed recombitron cultures after 3 rounds of editing. Three biological replicates are shown in open circles for each site, clustered over the number of recombitrons used.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Tables 1 and 3.

Reporting Summary

Supplementary Table 2

Recombitron plasmid details.

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Fishman, C.B., Crawford, K.D., Bhattarai-Kline, S. et al. Continuous multiplexed phage genome editing using recombitrons. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02370-5

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