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.

中文翻译：

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使用 CRISPR-Cas9 发现枯草芽孢杆菌噬菌体的必需和非必需基因时的机会和限制

毒性细菌病毒，也称为噬菌体或噬菌体，被认为是对抗抗生素耐药细菌的潜在选择。然而，它们的生物学仍然知之甚少，只有一小部分噬菌体基因被赋予了功能。为了实现第一次分类，我们探索了测试噬菌体基因对病毒复制的要求的新选择。作为模型，我们使用了已知最小的枯草芽孢杆菌噬菌体 Goe1 和基于 Cas9 的诱变载体 pRH030 作为遗传工具。所有噬菌体基因都被特异性破坏，并研究了个体存活率和突变基因型。通过宿主固有的非同源物末端连接系统或 Cas9 靶序列的自然改变，存活的噬菌体依赖于基因组完整性。噬菌体幸存者的量化和验证潜在的遗传情况能够对病毒复制的必需或非必需组中的基因进行分类。我们还观察到结构基因比基因组复制机制的基因拥有更多的自然突变。