Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis.
Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis.
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DOI：
10.1016/j.cels.2020.09.009
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发表时间：
2020-11-18
期刊：
影响因子：
9.3
通讯作者：
Gross CA
中科院分区：
文献类型：
--
作者：
Hawkins JS;Silvis MR;Koo BM;Peters JM;Osadnik H;Jost M;Hearne CC;Weissman JS;Todor H;Gross CA
必需基因是细胞网络的枢纽，但是缺乏用于滴定基因表达的高通量方法限制了我们对优化其表达水平的适应性景观的理解。我们开发了一种改良的CRISPRi系统，该系统利用不完全匹配的sgRNA的功效的可预测降低来产生确定水平的CRISPRi活性，并证明了其广泛的适用性。使用预测跨越全范围敲低水平的错配sgRNA文库，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，但在通路内是相似的。值得注意的是，尽管E. coli和B.枯草芽孢杆菌，最重要的同源物具有相似的表达适合度关系，具有罕见但信息丰富的差异。因此，必需基因的表达水平可能反映了两种生物体之间共享的稳态或进化约束。Hawkins和Silvis等人开发了一种通过将特异性错配引入CRISPRi sgRNA来可预测地滴定细菌中基因表达的系统。错配的sgRNA能够在单个实验中跨许多基因实现多个敲低水平。他们使用这种技术来确定大肠杆菌和枯草芽孢杆菌中所有必需基因的表达适应度曲线，发现它们在通路内和在相距约20亿年的同源物之间共享。
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. Hawkins and Silvis et al. develop a system for predictably titrating gene expression in bacteria by introducing specific mismatches into CRISPRi sgRNAs. Mismatched sgRNAs enable multiple knockdown levels across many genes in a single experiment. They use this technique to determine the expression-fitness curves of all essential genes in Escherichia coli and Bacillus subtilis, finding that they are shared within pathways and between homologs diverged by ~2 billion years.
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影响因子：
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