Institute of Biotechnology, BTU Cottbus-Senftenberg, Senftenberg, Germany.
Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark.
Methods Mol Biol. 2023;2555:205-212. doi: 10.1007/978-1-0716-2795-2_15.
Phages are viruses of bacteria and have been known for over a century. They do not have a metabolism or protein synthesis machinery and rely on host cells for replication. The model organism Bacillus subtilis has served as a host strain for decades and enabled the isolation of many unique viral strains. However, many viral species representatives remained orphans as no, or only a few, related phages were ever re-isolated.The presented protocol describes how a CRISPR-Cas9 system with an artificial CRISPR-array can be set up and used to discriminate abundant and well-known B. subtilis phage from a host-based metagenome enrichment. The obtained viral suspension can be used for metagenome sequencing and isolating new viral strains.
噬菌体是细菌病毒，其存在已有一个多世纪。它们没有新陈代谢或蛋白质合成机制，依赖宿主细胞进行复制。模式生物枯草芽孢杆菌作为宿主菌株已有数十年，可用于分离许多独特的病毒株。然而，许多病毒种代表仍然是孤儿，因为从未或仅重新分离出少数相关噬菌体。本方案描述了如何设置和使用带有人工 CRISPR 阵列的 CRISPR-Cas9 系统，以区分丰富且广为人知的枯草芽孢杆菌噬菌体与基于宿主的宏基因组富集物。获得的病毒悬浮液可用于宏基因组测序和分离新的病毒株。
