摘要

过度使用抗生素在耐多药细菌的出现和传播中起主要作用。针对细菌病原体的分子靶向特异性治疗方法可以通过降低微生物生长期间的选择压力来防止这个问题。在这里，我们介绍一种非病毒治疗策略，提供针对抗菌抗性的基因组编辑材料。我们将CRISPR-Cas9系统应用于运送货物，该系统已被公认为是一种用于在不同生物体中进行高度特异性和高效基因组工程的创新工具。我们利用聚合物衍生的Cas9，通过与阳离子聚合物直接共价修饰蛋白质，用于随后与靶向抗生素抗性的单引导RNA复合。我们展示纳米CRISPR复合物（= Cr-Nanocomplex）成功形成，同时保持Cas9核酸内切酶的功能活性，诱导双链DNA切割。我们还证明，设计用于靶向mecA（主要参与甲氧西林抗性的基因）的Cr-纳米复合物可以被有效地递送到耐甲氧西林金黄色葡萄球菌（MRSA）中，并且与使用相比能够以更高的效率编辑细菌基因组天然Cas9复合物或常规的基于脂质的制剂。本研究第一次显示共价修饰的CRISPR系统允许非病毒，治疗性基因组编辑，并且可以潜在地用作靶特异性抗微生物剂。

The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA—the major gene involved in methicillin resistance—can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.

https://pubs.acs.org/doi/abs/10.1021/acs.bioconjchem.6b00676