Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin.
Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, Wisconsin.
Curr Protoc Microbiol. 2020 Dec;59(1):e130. doi: 10.1002/cpmc.130.
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 the purpose 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. © 2020 The Authors. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning of new sgRNA spacers into Mobile-CRISPRi vectors Basic Protocol 3: Tn7 transfer of Mobile-CRISPRi to Gram-negative bacteria Basic Protocol 4: ICEBs1 transfer of Mobile-CRISPRi to Bacillales Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3: Transformation of E. coli by electroporation Support Protocol 4: Transformation of CaCl -competent E. coli.
简单的细菌基因组测序已经开启了一个真正的新型基因宝库，等待着功能探索。要充分利用这一机会，需要能够靶向各种细菌中所有基因的强大遗传工具。CRISPR 干扰 (CRISPRi) 是一种可编程的基因敲低工具，它使用由单个向导 RNA(sgRNA) 和一种无催化活性的 Cas9 核酸酶 (dCas9) 组成的 RNA-蛋白质复合物，来阻止靶基因的转录。我们之前开发了一套模块化的 CRISPRi 系统，可以通过共轭转移并整合到各种细菌的基因组中，我们称之为 Mobile-CRISPRi。在这里，我们提供了详细的协议，用于修饰和转移 Mobile-CRISPRi 载体，以敲低目标基因在感兴趣的细菌中的表达。我们进一步讨论了优化 Mobile-CRISPRi 敲低、转移和整合的策略。我们涵盖了以下基本协议：sgRNA 设计、将新的 sgRNA 间隔子克隆到 Mobile-CRISPRi 载体中、Tn7 转移 Mobile-CRISPRi 到革兰氏阴性菌、以及 ICEBs1 转移 Mobile-CRISPRi 到芽孢杆菌目。© 2020 作者。基本方案 1：sgRNA 设计基本方案 2：将新的 sgRNA 间隔子克隆到 Mobile-CRISPRi 载体中基本方案 3：Tn7 转移 Mobile-CRISPRi 到革兰氏阴性菌基本方案 4：ICEBs1 转移 Mobile-CRISPRi 到芽孢杆菌目支持方案 1：使用荧光报告基因定量 CRISPRi 抑制作用支持方案 2：使用平板 CRISPRi 点斑实验检测基因的必需性支持方案 3：通过电穿孔转化大肠杆菌支持方案 4：氯化钙感受态大肠杆菌的转化
