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RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering

Jingwen Guan, Agnès Oromí Bosch, Senén D. Mendoza, Shweta Karambelkar, Joel Berry, Joseph Bondy-Denomy
doi: https://doi.org/10.1101/2022.02.14.480438
Jingwen Guan
1Department of Microbiology & Immunology, University of California, San Francisco, San Francisco, CA, USA
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Agnès Oromí Bosch
2Felix Biotechnology, South San Francisco, CA, USA
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Senén D. Mendoza
1Department of Microbiology & Immunology, University of California, San Francisco, San Francisco, CA, USA
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Shweta Karambelkar
1Department of Microbiology & Immunology, University of California, San Francisco, San Francisco, CA, USA
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Joel Berry
2Felix Biotechnology, South San Francisco, CA, USA
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Joseph Bondy-Denomy
1Department of Microbiology & Immunology, University of California, San Francisco, San Francisco, CA, USA
3Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA, USA
4Innovative Genomics Institute, Berkeley, CA, USA
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  • For correspondence: joseph.bondy-denomy@ucsf.edu
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Abstract

The viruses that infect bacteria, bacteriophages (or phages), possess numerous genes of unknown function. Genetic tools are required to understand their biology and enhance their efficacy as antimicrobials. Pseudomonas aeruginosa jumbo phage ΦKZ and its relatives are a broad host range phage family that assemble a proteinaceous “phage nucleus” structure during infection. Due to the phage nucleus, DNA-targeting CRISPR-Cas is ineffective against this phage and thus there are currently no reverse genetic tools for this family. Here, we develop a DNA phage genome editing technology using the RNA-targeting CRISPR-Cas13a enzyme as a selection tool, an anti-CRISPR gene (acrVIA1) as a selectable marker, and homologous recombination. Precise insertion of foreign genes, gene deletions, and the addition of chromosomal fluorescent tags into the ΦKZ genome were achieved. Deletion of phuZ, which encodes a tubulin-like protein that centers the phage nucleus during infection, led to the mispositioning of the phage nucleus but surprisingly had no impact on phage replication, despite a proposed role in capsid trafficking. A chromosomal fluorescent tag placed on gp93, a proposed “inner body” protein in the phage head revealed a protein that is injected with the phage genome, localizes with the maturing phage nucleus, and is massively synthesized around the phage nucleus late in infection. Successful editing of two other phages that resist DNA-targeting CRISPR-Cas systems [OMKO1 (ΦKZ-like) and PaMx41] demonstrates the flexibility of this method. RNA-targeting Cas13a system holds great promise for becoming a universal genetic editing tool for intractable phages. This phage genetic engineering platform enables the systematic study of phage genes of unknown function and the precise modification of phages for use in a variety of applications.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted February 15, 2022.
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RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering
Jingwen Guan, Agnès Oromí Bosch, Senén D. Mendoza, Shweta Karambelkar, Joel Berry, Joseph Bondy-Denomy
bioRxiv 2022.02.14.480438; doi: https://doi.org/10.1101/2022.02.14.480438
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RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering
Jingwen Guan, Agnès Oromí Bosch, Senén D. Mendoza, Shweta Karambelkar, Joel Berry, Joseph Bondy-Denomy
bioRxiv 2022.02.14.480438; doi: https://doi.org/10.1101/2022.02.14.480438

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