Abstract
Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous ‘phage nucleus’ structure that protects from DNA-targeting CRISPR–Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR–Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the ФKZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR–Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function.
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Data availability
All relevant data are included in the paper and/or its supplementary information files. The complete genome sequence of OMKO1 was deposited in GenBank under accession no. ON631220. All strains and plasmids are available from the corresponding author upon reasonable request. Source data are provided with this paper.
Code availability
The data analysis code is available from public repositories at https://zenodo.org/record/6324407#.YiAjrejMI2w.
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Acknowledgements
The Bondy-Denomy lab was supported by the National Institutes of Health (no. R01GM127489 and R01AI171041), Vallee Foundation, Searle Scholarship, Innovative Genomics Institute and University of California San Francisco Program for Breakthrough Biomedical Research funded in part by the Sandler Foundation. This work was also supported by research funds from Felix Biotechnology. We thank L. Marraffini (The Rockefeller University) for providing the plasmid pAM383. We thank P. Turner and B. Chan (Yale University) for providing the OMKO1 phage and P. aeruginosa clinical isolates. We thank G. Guarneros Peña at Centro de Investigación y de Estudios Avanzados for providing the PaMx41 phage. We thank T. Rotstein for his generous assistance with NGS data processing and interpretation. We thank members of the Bondy-Denomy laboratory for productive conversations and generous suggestions for our work.
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Contributions
J.G. designed and performed the experiments, analysed the data and wrote the manuscript. A.O.-B. performed phage plaque and microplate liquid assays and analysed the data. S.D.M. designed and constructed the Cas13a crRNA vectors. S.K. conducted the phage plaque assays for phage PaMx41. J.B. performed NGS and analysed the data. J.B.-D. conceived and supervised the study, designed the experiments, acquired the funding and wrote the manuscript.
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J.B.-D. is a scientific advisory board member of SNIPR BIOME, Excision BioTherapeutics and Leapfrog Bio, and a scientific advisory board member and cofounder of Acrigen Biosciences. The Bondy-Denomy laboratory receives research support from Felix Biotechnology. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Plaque efficiency assays of distinct crRNAs of CRISPR-Cas13a targeting transcripts of diverse ΦKZ genes.
Ten-fold serial dilutions of ΦKZ spotted on lawns expressing cas13 and crRNAs targeting the indicated genes. The crRNA targeting orf120 highlighted in the red frame has been used for ΦKZ genome engineering. NT, non-targeting.
Extended Data Fig. 2 One-step growth curves of engineered ФKZ variants.
One-step growth curve experiment was performed to determine the latent time period and burst size of engineered phages. Representative plots are shown for each phage strain. The burst sizes are shown in brackets after of each ФKZ variant, representing the mean ± standard error of three or four biologically independent replicates.
Extended Data Fig. 3 Sequence alignment of wild type ΦKZ and three escape mutants at the engineered genomic site.
Escape mutants were isolated and verified by PCR and sequencing. The WT orf120 sequence is highlighted in blue and the downstream region is highlighted in grey. The stop codon (TGA) of orf120 is highlighted in green and Escape mutant #3 reconstitutes it to TAG. The sequence in the red frame matches the spacer sequence of the crRNA that was used to target and eliminate WT phages. Deletions were indicated by dashed lines and their corresponding numbers of absent base pairs.
Extended Data Fig. 4 Determination of host range of ΦKZ ∆phuZ and ∆orf93 mutants by plaque assay on P. aeruginosa clinical strains.
Spot-titration of the indicated ΦKZ phages on lawns of clinical isolates of P. aeruginosa (FB-XX).
Extended Data Fig. 5 Failure of genetic editing the shell gene (orf54) in ΦKZ.
(A) Schematic of genomes of WT ΦKZ and three mutated orf54 variants, “∆orf54”, “FP-orf54”, and “orf54-FP”, at the editing site. orf54, acrVIA1, and fluorescent protein (FP) are shown as blue, red, and green rectangles, respectively. F and R indicate forward and reverse primers, respectively, for PCR confirmation of orf54 engineering. (B) PCR confirmation of the indicated orf54 mutants using their corresponding pair of primers. All three mutants generated multiple bands, including a band in the same size as the single band produced by WT. PCR-based screening for engineered ΦKZ orf54 variants have been independently repeated at least three times yielding similar results. (C) Genome alignment of WT phage with the isolated orf54 “pseudo knock-out” mutant (“∆orf54”). A gene cluster of ~ 7 kbp (orf206 - orf216) was missing in the mutant, likely as a result of phage packaging capacity. The majority of the editing plasmid used to generate recombinants was at the editing site, leaving orf54 intact.
Extended Data Fig. 6 Host range assay of engineered OMKO1 variants.
Host ranges were determined by microplate liquid assay at MOI of 0.01 and 1 on 22 P. aeruginosa clinical strains. The values are presented as the mean liquid assay scores across three independent experiments. Asterisks (*) indicate significant difference between WT and engineered strains as determined by two-sided Students’ T-tests (p < 0.05). The color intensity of each phage-host combination reflects the liquid assay score, which represents how well the phage strain can repress the growth of a given bacterial host. No inhibition of bacterial growth is reflected by a liquid assay score of 0, and complete suppression would result in a score of 100.
Supplementary information
Supplementary Information
Supplementary Tables 1–4.
Supplementary Video 1
Time-lapse video of a P. aeruginosa cell infected by a ФKZ ΔphuZ mutant phage.
Supplementary Video 2
Time-lapse video of a P. aeruginosa cell infected by a ФKZ gp93-mNeonGreen mutant phage.
Supplementary Video 3
Time-lapse video of a P. aeruginosa cell infected by a wild-type ФKZ packaged with gp93-mNeonGreen fusion proteins in the capsid.
Source data
Source Data Fig. 1
Unprocessed agarose gels.
Source Data Fig. 2
Statistical source data.
Source Data Extended Data Fig. 3
Statistical source data.
Source Data Extended Data Fig. 5
Unprocessed agarose gels.
Source Data Extended Data Fig. 6
Statistical source data.
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Guan, J., Oromí-Bosch, A., Mendoza, S.D. et al. Bacteriophage genome engineering with CRISPR–Cas13a. Nat Microbiol 7, 1956–1966 (2022). https://doi.org/10.1038/s41564-022-01243-4
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DOI: https://doi.org/10.1038/s41564-022-01243-4
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