CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d

Adler, Benjamin A.; Al-Shimary, Muntathar J.; Patel, Jaymin R.; Armbruster, Emily G.; Colognori, David; Charles, Emeric J.; Miller, Kate V.; Lahiri, Arushi; Cui, Michael L.; Oromí-Bosch, Agnès; Voelker, Angela; Trinidad, Marena; Lee, Jina; Beurnier, Sebastien; Boger, Ron; Nomburg, Jason; Barrangou, Rodolphe; Mutalik, Vivek K.; Schoeniger, Joseph S.; Pogliano, Joseph A.; Savage, David F.; Doudna, Jennifer A.; Cress, Brady F.

doi:10.1038/s41564-025-01935-7

Title: CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d

Journal Article · 25 February 2025 · Nature Microbiology

DOI: https://doi.org/10.1038/s41564-025-01935-7 · OSTI ID:2551792

^[1]; Al-Shimary, Muntathar J. ^[1]; Patel, Jaymin R. ^[1];

^[2]; Colognori, David ^[1]; Charles, Emeric J. ^[1];

^[1]; Lahiri, Arushi ^[1]; Cui, Michael L. ^[1]; Oromí-Bosch, Agnès ^[1]; Voelker, Angela ^[1];

^[1]; Lee, Jina ^[2]; Beurnier, Sebastien ^[1];

^[1]; Nomburg, Jason ^[3];

^[4];

^[5]; Schoeniger, Joseph S. ^[6];

^[2] more »

University of California, Berkeley, CA (United States)
University of California, San Diego, CA (United States)
University of California, Berkeley, CA (United States); Gladstone Institute of Data Science and Biotechnology, San Francisco, CA (United States)
North Carolina State University, Raleigh, NC (United States)
University of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sandia National Laboratories (SNL-CA), Livermore, CA (United States)
University of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Sandia National Laboratories (SNL-CA), Livermore, CA (United States)

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.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: Leona M. and Harry B. Helmsley Charitable Trust; USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2551792

Journal Information:: Nature Microbiology, Journal Name: Nature Microbiology Journal Issue: 3 Vol. 10; ISSN 2058-5276

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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