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

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

Journal: Nature microbiology 2025;10(3):694-709

PMID: 40011704

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Abstract

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.

Address: California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA.; Innovative Genomics Institute, University of California, Berkeley, CA, USA.; Innovative Genomics Institute, University of California, Berkeley, CA, USA.; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.; Innovative Genomics Institute, University of California, Berkeley, CA, USA.; School of Biological Sciences, University of California, San Diego, La Jolla, CA, USA.; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA.; Graduate Group in Biophysics, University of California, Berkeley, CA, USA.; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA.; Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.; Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, USA.; Innovative Genomics Institute, University of California, Berkeley, CA, USA.; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.; Systems Biology Department, Sandia National Laboratories, Livermore, CA, USA.; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.; Howard Hughes Medical Institute, University of California, Berkeley, CA, USA.; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA. [email protected].; Innovative Genomics Institute, University of California, Berkeley, CA, USA. [email protected].; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. [email protected].; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. [email protected].; Systems Biology Department, Sandia National Laboratories, Livermore, CA, USA. [email protected].; Howard Hughes Medical Institute, University of California, Berkeley, CA, USA. [email protected].; Department of Chemistry, University of California, Berkeley, CA, USA. [email protected].; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. [email protected].; Innovative Genomics Institute, University of California, Berkeley, CA, USA. [email protected].

DOI: 10.1038/s41564-025-01935-7

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MeSH Terms: Bacteriophages, Genomics, CRISPR-Cas Systems, Genome, Viral, Phylogeny, RNA-Binding Proteins, Genetic Fitness, Bacteriophage lambda