Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli.
Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli.
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DOI：
10.1128/mbio.02561-21
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发表时间：
2021-10-26
期刊：
影响因子：
6.4
通讯作者：
Gross CA
中科院分区：
文献类型：
--
作者：
Silvis MR;Rajendram M;Shi H;Osadnik H;Gray AN;Cesar S;Peters JM;Hearne CC;Kumar P;Todor H;Huang KC;Gross CA
CRISPR干扰（CRISPRi）促进了使用高通量和靶向方法研究不同生物体中的必需基因。尽管这种技术有希望，但对于模型细菌大肠杆菌或任何革兰氏阴性菌，都不存在靶向必需基因的全面阵列CRISPRi文库。在这里，我们构建并描述了这样一个库。在我们的E.大肠杆菌文库含有靶向必需（或选择的非必需）基因的诱导型染色体整合的单向导RNA（sgRNA），并且可以与携带dcas9盒的假Hfr供体菌株交配以产生CRISPRi敲低菌株。使用该系统，我们构建了CRISPRi菌株的阵列库，并进行了群体和单细胞生长和形态测量以及有针对性的后续实验。这些研究发现，抑制翻译会导致延迟期延长，确定了新的细胞形态调节剂，并揭示了形态基因mreB受到转录反馈调节，这对维持形态至关重要。我们的研究结果突出了经典和非经典的作用，在细胞内稳态的许多方面的必要基因。
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.