Bhoobalan-Chitty Yuvaraj, Stouf Mathieu, De Paepe Marianne
Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France.
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
Front Genome Ed. 2024 Dec 19;6:1495968. doi: 10.3389/fgeed.2024.1495968. eCollection 2024.
CRISPR-Cas type II and type V systems are inefficient in modifying bacteriophage T4 genome, due to hypermodification of its DNA. Here, we present a genome editing technique for bacteriophage T4 using the type VI CRISPR-Cas system. Using BzCas13b targeting of T4 phage, we were able to individually delete both T4 glucosyl transferase genes, and . Furthermore, we employed this method to mutate two conserved residues within the T4 DNA polymerase and to introduce the yellow fluorescent protein (YFP) coding sequence into T4 phage genome, enabling us to visualize phage infections. This T4 genome editing protocol was optimized to generate recombinant phages within a 6-hour timescale. Finally, spacers homologous to a variety of T4 genes were used to study the generality of Cas13b targeting, revealing important variability in targeting efficiency. Overall, this method constitutes a rapid and effective means of generating specific T4 phage mutants, which could be extended to other T4-like phages.
由于噬菌体T4的DNA高度甲基化，CRISPR-Cas II型和V型系统在修饰噬菌体T4基因组方面效率低下。在此，我们展示了一种使用VI型CRISPR-Cas系统对噬菌体T4进行基因组编辑的技术。通过使用靶向T4噬菌体的BzCas13b，我们能够分别删除T4葡糖基转移酶基因和。此外，我们采用此方法对T4 DNA聚合酶内的两个保守残基进行突变，并将黄色荧光蛋白（YFP）编码序列引入T4噬菌体基因组，从而使我们能够观察噬菌体感染。此T4基因组编辑方案经过优化，可在6小时内产生重组噬菌体。最后，使用与多种T4基因同源的间隔序列来研究Cas13b靶向的普遍性，揭示了靶向效率的重要变异性。总体而言，该方法构成了一种快速有效的产生特定T4噬菌体突变体的手段，可扩展至其他T4样噬菌体。
