Abstract：
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has enabled rapid advances in genomic engineering and transcriptional regulation. Specifically, CRISPR interference (CRISPRi) system has been used to systematically investigate the gene functions of microbial strains in a high-throughput manner. This method involves growth profiling using cells that have been transformed with the deactivated Cas9 (dCas9) and single-guide RNA (sgRNA) libraries that target individual genes. The fitness scores of each gene are calculated by measuring the abundance of individual sgRNAs during cell growth and represent gene essentiality. In this chapter, a process is described for functional genetic screening using CRISPRi at the whole-genome scale, starting from the synthesis of sgRNA libraries, construction of CRISPRi libraries, and identification of essential genes through growth profiling. The commensal bacterium Bacteroides thetaiotaomicron was used to implement the protocol. This method is expected to be applicable to a broader range of microorganisms to explore the novel phenotypic characteristics of microorganisms.
摘要：
聚集的定期间隔短回文重复(CRISPR)/Cas9系统使得基因组工程和转录调控的快速进展成为可能。具体来说,CRISPR干扰（CRISPRi）系统已用于以高通量方式系统地研究微生物菌株的基因功能。该方法涉及使用已经用靶向单个基因的失活Cas9(dCas9)和单向导RNA(sgRNA)文库转化的细胞进行生长谱分析。每个基因的适应度评分是通过测量细胞生长过程中单个sgRNA的丰度来计算的，代表基因的重要性。在这一章中,描述了使用CRISPRi在全基因组尺度上进行功能遗传筛选的过程，从sgRNA文库的合成开始，CRISPRI图书馆的建设，并通过生长谱分析鉴定必需基因。共生细菌拟杆菌属细菌用于实施该方案。该方法有望适用于更广泛的微生物，以探索微生物的新表型特征。
