| Autor: |
Silvis MR; Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California, USA., Rajendram M; Department of Bioengineering, Stanford Universitygrid.168010.e, Stanford, California, USA., Shi H; Department of Microbiology and Immunology, Stanford Universitygrid.168010.e School of Medicine, Stanford, California, USA., Osadnik H; Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California, USA., Gray AN; Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California, USA., Cesar S; Department of Microbiology and Immunology, Stanford Universitygrid.168010.e School of Medicine, Stanford, California, USA., Peters JM; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA., Hearne CC; Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California, USA., Kumar P; Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California, USA., Todor H; Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California, USA., Huang KC; Department of Bioengineering, Stanford Universitygrid.168010.e, Stanford, California, USA.; Department of Microbiology and Immunology, Stanford Universitygrid.168010.e School of Medicine, Stanford, California, USA.; Chan Zuckerberg Biohub, San Francisco, California, USA., Gross CA; Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California, USA. |
| Abstrakt: |
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. IMPORTANCE 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. |
