School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, U.K.
Department of Infection Biology, London School of Hygiene & Tropical Medicine, London WC1E 7HT, U.K.
ACS Synth Biol. 2023 Mar 17;12(3):709-721. doi: 10.1021/acssynbio.2c00465. Epub 2023 Feb 20.
The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections is hampered by the inefficient delivery of 9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into and the dysentery-causing to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site () significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting phagemids into using a zebrafish larvae infection model, where they significantly reduce the bacterial load and promote host survival. Our study highlights the potential of combining P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.
簇状、规则间隔、短回文重复序列 (CRISPR) 和 Cas9 RNA 指导的核酸酶的发现为有选择地杀死特定的细菌种群或物种提供了前所未有的机会。然而，由于将 9 个基因构建体有效递送至细菌细胞的效率低下，CRISPR-Cas9 用于清除细菌感染受到了阻碍。在这里，我们使用一种广谱宿主范围的 P1 衍生噬菌体来递送 CRISPR-Cas9 染色体靶向系统进入 和引起痢疾的 ，以实现对靶向细菌细胞的 DNA 序列特异性杀伤。我们表明，对辅助 P1 噬菌体 DNA 包装位点（）的遗传修饰显著提高了包装噬菌体的纯度，并提高了 Cas9 介导的 细胞杀伤效果。我们进一步证明，P1 噬菌体颗粒可以使用斑马鱼幼虫感染模型将靶向染色体的 噬菌体递送至 ，从而显著降低细菌负荷并促进宿主存活。我们的研究强调了将 P1 噬菌体为基础的递呈与 CRISPR 染色体靶向系统相结合以实现 DNA 序列特异性细胞致死性和有效清除细菌感染的潜力。
