Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli
Melanie R. Silvis, Manohary Rajendram, Handuo Shi, Hendrik Osadnik, Andrew N. Gray, Spencer Cesar, Jason M. Peters, Cameron C. Hearne, Parth Kumar, Horia Todor, Kerwyn Casey Huang, Carol A. Gross
Abstract
Essential genes make up only ∼5 to 10% of the genetic complement in most organisms but occupy much of their protein synthesis and account for almost all antibiotic targets. Despite the importance of essential genes, their intractability has, until recently, hampered efforts to study them. CRISPRi has facilitated the study of essential genes by allowing inducible and titratable depletion. However, all large-scale CRISPRi studies in Gram-negative bacteria thus far have used plasmids to express CRISPRi components and have been constructed in pools, limiting their utility for targeted assays and complicating the determination of antibiotic effects. Here, we use a modular method to construct an arrayed library of chromosomally integrated CRISPRi strains targeting the essential genes of the model bacterium Escherichia coli. This library enables targeted studies of essential gene depletions and high-throughput determination of antibiotic targets and facilitates studies targeting the outer membrane, an essential component that serves as the major barrier to antibiotics.