美国哥伦比亚大学Saeed Tavazoie课题组通过CRISPR适应的全基因组扰动，揭示了抗生素敏感性的复杂遗传学。论文发表于2020年2月27日的《细胞》。

研究人员使用化脓性链球菌CRISPR-Cas适应机制开发了CRISPR适应介导的文库加工（CALM），它将细菌细胞转变为“工厂”，以产生覆盖95％所有可靶向基因组位点的数十万个crRNA。这些高度全面的CRISPRi文库的基因平均被100多种不同的crRNA靶向，可产生不同程度的转录抑制，这对于揭示新型抗生素抗性决定簇至关重要。

此外，通过迭代CRISPR适应，他们快速生成了代表超过100,000个双基因扰动的双crRNA文库。间隔物适应性的极化性质揭示了逐步获取遗传扰动导致抗生素抗性增加的历史偶然性。CALM避免了gRNA合成和克隆所需的费用、劳力和时间，从而允许在常规基因操作难以完成的野生型细菌中生成CRISPRi文库。

据介绍，全基因组的CRISPR筛选可以对基因功能进行系统的询问。然而，指导RNA文库的合成成本高昂，并且其有限的多样性减弱了CRISPR筛选的敏感性。

附：英文原文

Title: Comprehensive Genome-wide Perturbations via CRISPR Adaptation Reveal Complex Genetics of Antibiotic Sensitivity

Author: Wenyan Jiang, Panos Oikonomou, Saeed Tavazoie

Issue&Volume: 2020-02-27

Abstract: Genome-wide CRISPR screens enable systematic interrogation of gene function. However,guide RNA libraries are costly to synthesize, and their limited diversity compromisesthe sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR adaptation-mediated librarymanufacturing (CALM), which turns bacterial cells into “factories” for generatinghundreds of thousands of crRNAs covering 95% of all targetable genomic sites. Withan average gene targeted by more than 100 distinct crRNAs, these highly comprehensiveCRISPRi libraries produced varying degrees of transcriptional repression criticalfor uncovering novel antibiotic resistance determinants. Furthermore, by iteratingCRISPR adaptation, we rapidly generated dual-crRNA libraries representing more than100,000 dual-gene perturbations. The polarized nature of spacer adaptation revealedthe historical contingency in the stepwise acquisition of genetic perturbations leadingto increasing antibiotic resistance. CALM circumvents the expense, labor, and timerequired for synthesis and cloning of gRNAs, allowing generation of CRISPRi librariesin wild-type bacteria refractory to routine genetic manipulation.

DOI: 10.1016/j.cell.2020.02.007

Source: https://www.cell.com/cell/fulltext/S0092-8674(20)30151-3