<br>P1 噬菌体实现 CRISPR-Cas9 对福氏志贺氏菌的抗菌活性,ACS Synthetic Biology

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P1 噬菌体实现 CRISPR-Cas9 对福氏志贺氏菌的抗菌活性
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2023-02-20 , DOI: 10.1021/acssynbio.2c00465
Yang W Huan ₁ , Vincenzo Torraca _{2,

3} , Russell Brown ₁ , Jidapha Fa-Arun ₁ , Sydney L Miles ₂ , Diego A Oyarzún _{1,

4} , Serge Mostowy ₂ , Baojun Wang _{5,

6}

Affiliation

成簇、规则间隔、短回文重复序列 (CRISPR) 和 Cas9 RNA 引导核酸酶的发现为选择性杀死特定细菌群体或物种提供了前所未有的机会。然而，使用 CRISPR-Cas9 清除体内细菌感染受到cas 9 基因构建体向细菌细胞传递效率低下的阻碍。在这里，我们使用广泛宿主范围的 P1 衍生噬菌粒将 CRISPR-Cas9 染色体靶向系统传递到大肠杆菌和引起痢疾的福氏志贺氏菌中，以实现对目标细菌细胞的 DNA 序列特异性杀伤。我们发现，辅助 P1 噬菌体 DNA 包装位点 ( pac ) 的基因修饰显着提高了包装噬菌粒的纯度，并改善了 Cas9 介导的福氏链球菌细胞杀伤能力。我们进一步证明，使用斑马鱼幼虫感染模型，P1 噬菌体颗粒可以将染色体靶向的cas9噬菌粒递送到福氏链球菌体内，显着降低细菌负荷并促进宿主存活。我们的研究强调了将基于 P1 噬菌体的递送与 CRISPR 染色体靶向系统相结合以实现 DNA 序列特异性细胞致死和有效清除细菌感染的潜力。

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P1 Bacteriophage-Enabled Delivery of CRISPR-Cas9 Antimicrobial Activity Against Shigella flexneri

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 in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneriin vivo 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.

更新日期：2023-02-20

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