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Targeted genetic screening in bacteria with a Cas12k-guided transposase

Cell Rep. 2021-08; 
Weizhong Chen, Ze-Hui Ren, Na Tang, Guoshi Chai, Hongyuan Zhang, Yifei Zhang, Jiacheng Ma, Zhaowei Wu, Xia Shen, Xingxu Huang, Guan-Zheng Luo, Quanjiang Ji
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PCR Cloning and Subcloning … The designed oligonucleotide library was synthesized on a chip by Genscript (Nanjing, China). … The synthesized oligonucleotide pool was PCR-amplified and the PCR products were assembled into the pGTPA plasmid via GenBuilder™ Plus Cloning kit (Genscript, Nanjing, … Get A Quote

摘要

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 aerugin... More

关键词

CRISPR-Cas12k, Pseudomonas aeruginosa, genome engineering, high-throughput, library construction, phenotype screening, site-specific, transcription factor, transposition