计算生物学
生物
基因组
瓶颈
管道(软件)
协议(科学)
转座子突变
计算机科学
基因
遗传学
转座因子
医学
病理
嵌入式系统
程序设计语言
替代医学
作者
Vincent de Bakker,Xue Liu,Afonso M. Bravo,Jan‐Willem Veening
出处
期刊:Nature Protocols
[Springer Nature]
日期:2022-01-07
卷期号:17 (2): 252-281
被引量:43
标识
DOI:10.1038/s41596-021-00639-6
摘要
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.
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