Application of combined CRISPR screening for genetic and chemical-genetic interaction profiling in Mycobacterium tuberculosis.
Application of combined CRISPR screening for genetic and chemical-genetic interaction profiling in Mycobacterium tuberculosis.
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DOI：
10.1126/sciadv.add5907
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发表时间：
2022-11-25
期刊：
影响因子：
13.6
通讯作者：
中科院分区：
文献类型：
--
作者：
CRISPR筛选，包括CRISPR干扰（CRISPRi）和CRISPR-knockout（CRISPR-KO）筛选，已成为真核生物基因筛选中的有力技术。与真核生物相比，CRISPR-KO筛选尚未应用于细菌的功能基因组学研究。在这里，我们在结核分枝杆菌（Mtb）中构建了基因组规模的CRISPR-KO和CRISPRi文库。我们首先检查了这些文库以鉴定Mtb生存力所必需的基因。随后的筛选确定了数十个与抗结核药物贝达喹啉（BDQ）的耐药/易感性相关的基因。筛选结果的遗传和化学验证表明，它提供了一个有价值的资源，以调查的作用机制BDQ的影响，并确定化学遗传协同作用，可用于优化结核病治疗。总之，我们的研究结果证明了在细菌中进行有效的全基因组CRISPR-KO筛选的潜力，并建立了一种组合的CRISPR筛选方法，用于Mtb中遗传和化学-遗传相互作用的高通量研究。联合CRISPRi和CRISPR-KO筛选的开发为结核分枝杆菌提供了全面的功能基因组学研究平台。
CRISPR screening, including CRISPR interference (CRISPRi) and CRISPR-knockout (CRISPR-KO) screening, has become a powerful technology in the genetic screening of eukaryotes. In contrast with eukaryotes, CRISPR-KO screening has not yet been applied to functional genomics studies in bacteria. Here, we constructed genome-scale CRISPR-KO and also CRISPRi libraries in Mycobacterium tuberculosis (Mtb). We first examined these libraries to identify genes essential for Mtb viability. Subsequent screening identified dozens of genes associated with resistance/susceptibility to the antitubercular drug bedaquiline (BDQ). Genetic and chemical validation of the screening results suggested that it provided a valuable resource to investigate mechanisms of action underlying the effects of BDQ and to identify chemical-genetic synergies that can be used to optimize tuberculosis therapy. In summary, our results demonstrate the potential for efficient genome-wide CRISPR-KO screening in bacteria and establish a combined CRISPR screening approach for high-throughput investigation of genetic and chemical-genetic interactions in Mtb. Development of combined CRISPRi and CRISPR-KO screening provides a comprehensive functional genomics study platform in Mtb.
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影响因子：
6.4
作者：
DeJesus MA;Gerrick ER;Xu W;Park SW;Long JE;Boutte CC;Rubin EJ;Schnappinger D;Ehrt S;Fortune SM;Sassetti CM;Ioerger TR
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