School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China.
MOE Key Laboratory of Gene Function and Regulation, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China.
Cell Rep. 2021 Aug 31;36(9):109635. doi: 10.1016/j.celrep.2021.109635.
Microbes employ sophisticated cellular networks encoded by complex genomes to rapidly adapt to changing environments. High-throughput genome engineering methods are valuable tools for functionally profiling genotype-phenotype relationships and understanding the complexity of cellular networks. However, current methods either rely on special homologous recombination systems and are thus applicable in only limited bacterial species or can generate only nonspecific mutations and thus require extensive subsequent screening. Here, we report a site-specific transposon-assisted genome engineering (STAGE) method that allows high-throughput Cas12k-guided mutagenesis in various microorganisms, such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Exploiting the powerful STAGE technique, we construct a site-specific transposon mutant library that focuses on all possible transcription factors (TFs) in P. aeruginosa, enabling the comprehensive identification of essential genes and antibiotic-resistance-related factors. Given its broad host range activity and easy programmability, this method can be widely adapted to diverse microbial species for rapid genome engineering and strain evolution.
微生物利用复杂基因组编码的复杂细胞网络来快速适应不断变化的环境。高通量基因组工程方法是功能分析基因型-表型关系和理解细胞网络复杂性的有价值的工具。然而，目前的方法要么依赖于特殊的同源重组系统，因此只能在有限的细菌物种中应用，要么只能产生非特异性突变，因此需要广泛的后续筛选。在这里，我们报告了一种基于转座子辅助的基因组工程（STAGE）的方法，它允许在各种微生物中进行高通量 Cas12k 引导的诱变，如铜绿假单胞菌和肺炎克雷伯菌。利用强大的 STAGE 技术，我们构建了一个专注于铜绿假单胞菌中所有可能的转录因子（TFs）的定点转座子突变体文库，从而能够全面鉴定必需基因和抗生素抗性相关因子。鉴于其广泛的宿主范围活性和易于编程性，该方法可以广泛应用于不同的微生物物种，用于快速基因组工程和菌株进化。
