PDF(Pubmed)
Abstract：
Advances in gene editing, in particular CRISPR interference (CRISPRi), have enabled depletion of essential cellular machinery to study the downstream effects on bacterial physiology. Here, we describe the construction of an ordered E. coli CRISPRi collection, designed to knock down the expression of 356 essential genes with the induction of a catalytically inactive Cas9, harbored on the conjugative plasmid pFD152. This mobile CRISPRi library can be conjugated into other ordered genetic libraries to assess combined effects of essential gene knockdowns with non-essential gene deletions. As proof of concept, we probed cell envelope synthesis with two complementary crosses: (1) an Lpp deletion into every CRISPRi knockdown strain and (2) the lolA knockdown plasmid into the Keio collection. These experiments revealed a number of notable genetic interactions for the essential phenotype probed and, in particular, showed suppressing interactions for the loci in question.
摘要：
基因编辑的进展,特别是CRISPR干扰（CRISPRi），已经使必需的细胞机制耗尽，以研究对细菌生理学的下游影响。这里,我们描述了一个有序的大肠杆菌CRISPRi集合的构建，设计用于通过诱导具有催化活性的Cas9来敲除356个必需基因的表达，该Cas9包含在接合质粒pFD152上。该移动CRISPRi文库可以缀合到其他有序的遗传文库中,以评估必需基因敲除与非必需基因缺失的组合效应。作为概念的证明，我们用两个互补杂交来探测细胞包膜合成:(1)每个CRISPRi敲低菌株中的Lpp缺失和(2)Keio集合中的lolA敲低质粒。这些实验揭示了所探测的基本表型的许多显著的遗传相互作用，特别是,显示抑制所讨论基因座的相互作用。
