Laboratory of Gene Technology, Department of Biosystems, KU Leuven, Leuven, Belgium.
Methods Mol Biol. 2024;2793:113-128. doi: 10.1007/978-1-0716-3798-2_8.
The vast number of unknown phage-encoded ORFan genes and limited insights into the genome organization of phages illustrate the need for efficient genome engineering tools to study bacteriophage genes in their natural context. In addition, there is an application-driven desire to alter phage properties, which is hampered by time constraints for phage genome engineering in the bacterial host. We here describe an optimized CRISPR-Cas3 system in Pseudomonas for straightforward editing of the genome of virulent bacteriophages. The two-vector system combines a broad host range CRISPR-Cas3 targeting plasmid with a SEVA plasmid for homologous directed repair, which enables the creation of clean deletions, insertions, or substitutions in the phage genome within a week. After creating the two plasmids separately, a co-transformation to P. aeruginosa cells is performed. A subsequent infection with the targeted phage allows the CRISPR-Cas3 system to cut the DNA specifically and facilitate or select for homologous recombination. This system has also been successfully applied for P. aeruginosa and Pseudomonas putida genome engineering. The method is straightforward, efficient, and universal, enabling to extrapolate the system to other phage-host pairs.
大量未知噬菌体编码的 ORFan 基因和对噬菌体基因组组织的有限了解表明，需要有效的基因组工程工具来研究噬菌体基因在其自然环境中的作用。此外，人们出于应用的需求希望改变噬菌体的特性，但由于在细菌宿主中进行噬菌体基因组工程的时间限制，这一需求受到了阻碍。在这里，我们描述了一种在铜绿假单胞菌中优化的 CRISPR-Cas3 系统，用于简单地编辑毒性噬菌体的基因组。该双载体系统结合了一个广谱宿主范围的靶向 CRISPR-Cas3 的质粒和一个 SEVA 质粒，用于同源定向修复，这使得在一周内就能在噬菌体基因组中创建干净的缺失、插入或替换。在分别创建了这两个质粒后，将它们共同转化到铜绿假单胞菌细胞中。随后用靶向噬菌体感染，允许 CRISPR-Cas3 系统特异性地切割 DNA，并促进或选择同源重组。该系统也已成功应用于铜绿假单胞菌和恶臭假单胞菌的基因组工程。该方法简单、高效且具有通用性，可将该系统推广到其他噬菌体-宿主对。
