Virulent bacterial viruses, also known as phages or bacteriophages, are considered as a potential option to fight antibiotic-resistant bacteria. However, their biology is still poorly understood, and only a fraction of phage genes is assigned with a function. To enable the first classification, we explored new options to test phage genes for their requirement on viral replication. As a model, we used the smallest known Bacillus subtilis phage Goe1, and the Cas9-based mutagenesis vector pRH030 as a genetic tool. All phage genes were specifically disrupted, and individual survival rates and mutant genotypes were investigated. Surviving phages relied on the genome integrity through host intrinsic non-homologues end joining system or a natural alteration of the Cas9 target sequence. Quantification of phage survivors and verifying the underlying genetic situation enables the classification of genes in essential or non-essential sets for viral replication. We also observed structural genes to hold more natural mutations than genes of the genome replication machinery.
CITATION STYLE
Kohm, K., Basu, S., Nawaz, M. M., & Hertel, R. (2021). Chances and limitations when uncovering essential and non-essential genes of Bacillus subtilis phages with CRISPR-Cas9. Environmental Microbiology Reports, 13(6), 934–944. https://doi.org/10.1111/1758-2229.13005
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