CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d
Bacteriophages constitute one of the largest reservoirs of genes of unknown function in the biosphere. Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because phages subvert many modern functional genomics tools. Here we leverage RNA-targeting dCas13d to selectively interfere with protein translation and to measure phage gene fitness at a transcriptome-wide scale. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective across phage phylogeny, from model ssRNA, ssDNA and dsDNA phages to nucleus-forming jumbo phages. Using CRISPRi-ART, we determine a conserved role of diverse rII homologues in subverting phage Lambda RexAB-mediated immunity to superinfection and identify genes critical for phage fitness. CRISPRi-ART establishes a broad-spectrum phage functional genomics platform, revealing more than 90 previously unknown genes important for phage fitness.
Authors
- Jina Lee (ORCID: https://orcid.org/0000-0002-7808-1045)
- Joe Pogliano (ORCID: https://orcid.org/0000-0001-8989-2655)
- Arushi Lahiri (ORCID: https://orcid.org/0000-0001-6834-4692)
- Jason Nomburg (ORCID: https://orcid.org/0000-0001-7807-8658)
- David F. Savage (ORCID: https://orcid.org/0000-0003-0042-2257)
- David Colognori (ORCID: https://orcid.org/0000-0001-6995-7079)
- Emeric Charles (ORCID: https://orcid.org/0000-0002-3465-5625)
- Vivek K. Mutalik (ORCID: https://orcid.org/0000-0001-7934-0400)
- Brady F. Cress (ORCID: https://orcid.org/0000-0002-2948-2846)
- Marena Trinidad (ORCID: https://orcid.org/0000-0001-7839-4642)
- Jaymin R. Patel (ORCID: https://orcid.org/0000-0002-4380-4805)
- Michael Cui (ORCID: https://orcid.org/0000-0002-4352-9831)
- A Voelker
- Emily G. Armbruster (ORCID: https://orcid.org/0000-0003-0377-1209)
- Jennifer A. Doudna (ORCID: https://orcid.org/0000-0001-9161-999X)
- Joseph S. Schoeniger (ORCID: https://orcid.org/0000-0002-2719-7435)
- Rodolphe Barrangou (ORCID: https://orcid.org/0000-0002-0648-3504)
- Agnès Oromí-Bosch
- Benjamin A. Adler (ORCID: https://orcid.org/0000-0002-7488-3040)
- Muntathar Al-Shimary (ORCID: https://orcid.org/0000-0002-7021-2565)
- Sebastien Beurnier (ORCID: https://orcid.org/0000-0003-1533-8787)
- Ron Boger (ORCID: https://orcid.org/0000-0002-4467-271X)
- Kate V. Miller (ORCID: https://orcid.org/0000-0002-5315-4172)
Institutions
- QB3 (US)
- Gladstone Institutes (US)
- Howard Hughes Medical Institute (US)
- North Carolina State University (US)
- Lawrence Berkeley National Laboratory (US)
- Sandia National Laboratories California (US)
- University of California San Diego (US)
- Innovative Genomics Institute (US)
- University of California, Berkeley (US)
Publication Details
- Journal
- Nature Microbiology
- Published
- 2025-02-26
- DOI
- https://doi.org/10.1038/s41564-025-01935-7
- Citations
- 21
- Primary Topic
- Bacteriophages and microbial interactions
- Type
- article
- Field-Weighted Citation Impact
- 36.04
Funders
- National Science Foundation
- U.S. Department of Energy
- Silicon Valley Community Foundation
- Leona M. and Harry B. Helmsley Charitable Trust
- Innovative Genomics Institute
- Emerson Collective
- Valhalla Foundation
- Office of Science
- Biological and Environmental Research
- Lawrence Berkeley National Laboratory