Chances and limitations when uncovering essential and non-essential genes of Bacillus subtilis phages with CRISPR-Cas9

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Abstract

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.

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APA

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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