Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94158, USA.
Cell Syst. 2020 Nov 18;11(5):523-535.e9. doi: 10.1016/j.cels.2020.09.009. Epub 2020 Oct 19.
Essential genes are the hubs of cellular networks, but lack of high-throughput methods for titrating gene expression has limited our understanding of the fitness landscapes against which their expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate defined levels of CRISPRi activity and demonstrated its broad applicability. Using libraries of mismatched sgRNAs predicted to span the full range of knockdown levels, we characterized the expression-fitness relationships of most essential genes in Escherichia coli and Bacillus subtilis. We find that these relationships vary widely from linear to bimodal but are similar within pathways. Notably, despite ∼2 billion years of evolutionary separation between E. coli and B. subtilis, most essential homologs have similar expression-fitness relationships with rare but informative differences. Thus, the expression levels of essential genes may reflect homeostatic or evolutionary constraints shared between the two organisms.
必需基因是细胞网络的枢纽，但缺乏高通量的基因表达滴定方法限制了我们对其表达水平优化的适应景观的理解。我们开发了一种改良的 CRISPRi 系统，利用不完全匹配的 sgRNA 功效降低的可预测性来产生定义水平的 CRISPRi 活性，并证明了其广泛的适用性。使用预测的错配 sgRNA 文库，这些 sgRNA 预计会跨越全范围的敲低水平，我们表征了大肠杆菌和枯草芽孢杆菌中大多数必需基因的表达-适应性关系。我们发现这些关系从线性到双峰变化很大，但在途径内相似。值得注意的是，尽管大肠杆菌和枯草芽孢杆菌之间有大约 20 亿年的进化分离，但大多数必需的同源物具有相似的表达-适应性关系，只有很少但有信息的差异。因此，必需基因的表达水平可能反映了两个生物体之间共享的内稳态或进化限制。
