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Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

机译：可编程基因击倒在不同的细菌使用移动CRISPRi应承担的

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摘要

Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for thepurpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICEBs1 transfer of Mobile‐CRISPRi to Bacillales.
机译：灵巧的细菌基因组测序已解锁名副其实的新型基因宝库等待功能的探索。机会需要强大的遗传工具可以针对所有的基因在不同的细菌。干扰(CRISPRi)是一种可编程基因检测可拆卸的工具,它使用一个RNA蛋白质复杂的组成一个指南的RNA (sgRNA)和催化地活动Cas9核酸酶(dCas9) sterically阻止转录的目标基因。模块化CRISPRi系统转移结合和集成的基因组不同的细菌,我们称之为移动CRISPRi。在这里,我们提供详细的协议的修改和传输的移动CRISPRi向量击倒目标的目的感兴趣的基因的细菌。策略优化移动CRISPRi击倒、转移和整合。下列基本协议:sgRNA设计,克隆新的sgRNA间距器移动CRISPRi向量,Tn7移动必经CRISPRi转移革兰氏阴性细菌应承担和ICEBs1转让移动量CRISPRi Bacillales。

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