Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland.
Guangdong Key Laboratory for Genome Stability and Human Disease Prevention, Department of Pharmacology, International Cancer Center, Shenzhen University Health Science Center, Shenzhen, China.
Nat Protoc. 2022 Feb;17(2):252-281. doi: 10.1038/s41596-021-00639-6. Epub 2022 Jan 7.
CRISPR interference (CRISPRi) is a powerful tool to link essential and nonessential genes to specific phenotypes and to explore their functions. Here we describe a protocol for CRISPRi screenings to assess genome-wide gene fitness in a single sequencing step (CRISPRi-seq). We demonstrate the use of the protocol in Streptococcus pneumoniae, an important human pathogen; however, the protocol can easily be adapted for use in other organisms. The protocol includes a pipeline for single-guide RNA library design, workflows for pooled CRISPRi library construction, growth assays and sequencing steps, a read analysis tool (2FAST2Q) and instructions for fitness quantification. We describe how to make an IPTG-inducible system with small libraries that are easy to handle and cost-effective and overcome bottleneck issues, which can be a problem when using similar, transposon mutagenesis-based methods. Ultimately, the procedure yields a fitness score per single-guide RNA target for any given growth condition. A genome-wide screening can be finished in 1 week with a constructed library. Data analysis and follow-up confirmation experiments can be completed in another 2-3 weeks.
CRISPR 干扰（CRISPRi）是将必需基因和非必需基因与特定表型联系起来并探索其功能的强大工具。在这里，我们描述了一种用于 CRISPRi 筛选的方案，可在单个测序步骤中评估全基因组基因适应性（CRISPRi-seq）。我们展示了该方案在肺炎链球菌中的应用，肺炎链球菌是一种重要的人类病原体；但是，该方案可以轻松地适应于其他生物体。该方案包括一个单指导 RNA 文库设计的流水线、用于池式 CRISPRi 文库构建、生长测定和测序步骤的工作流程、一个读分析工具（2FAST2Q）和适应性定量的说明。我们描述了如何制作具有小文库的 IPTG 诱导系统，该系统易于操作且具有成本效益，并且克服了类似的基于转座子诱变的方法可能存在的瓶颈问题。最终，该程序为任何给定的生长条件的每个单指导 RNA 靶标提供一个适应性得分。一个全基因组筛选可以在构建文库后的 1 周内完成。数据分析和后续确认实验可以在另外 2-3 周内完成。
