Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli

CRISPR interference (CRISPRi) has facilitated the study of essential genes in diverse organisms using both high-throughput and targeted approaches. Despite the promise of this technique, no comprehensive arrayed CRISPRi library targeting essential genes exists for the model bacterium Escherichia coli , or for any Gram-negative species. Here, we built and characterized such a library. Each of the ∼500 strains in our E. coli library contains an inducible, chromosomally integrated single guide RNA (sgRNA) targeting an essential (or selected nonessential) gene and can be mated with a pseudo-Hfr donor strain carrying a dcas9 cassette to create a CRISPRi knockdown strain. Using this system, we built an arrayed library of CRISPRi strains and performed population and single-cell growth and morphology measurements as well as targeted follow-up experiments. These studies found that inhibiting translation causes an extended lag phase, identified new modulators of cell morphology, and revealed that the morphogene mreB is subject to transcriptional feedback regulation, which is critical for the maintenance of morphology. Our findings highlight canonical and noncanonical roles for essential genes in numerous aspects of cellular homeostasis.