CRISPR-dCpf1 mediated whole genome crRNA inhibition library for high-throughput screening of growth characteristic genes in Bacillus amyloliquefaciens LB1ba02.

Bacillus amyloliquefaciens LB1ba02 is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. However, autolysis affects the growth of bacteria, further affecting the yield of target products. Besides, the restriction-modification system, existed in B. amyloliquefaciens LB1ba02, results in a low transformation efficiency, which further leads to a lack of high-throughput screening tools. Here, we constructed a genome-wide crRNA inhibition library based on the CRISPR/dCpf1 system and high-throughput screening of related genes affecting the cell growth and autolysis using flow cytometry in B. amyloliquefaciens LB1ba02. The whole genome crRNA library was first validated for resistance to the toxic chemical 5-fluorouracil, and then used for validation of essential genes. In addition, seven gene loci (oppD, flil, tuaA, prmA, sigO, hslU, and GE03231) that affect the growth characteristics of LB1ba02 were screened. Among them, the Opp system had the greatest impact on growth. When the expression of operon oppA-oppB-oppC-oppD-oppF was inhibited, the cell growth difference was most significant. Inhibition of other sites could also promote rapid growth of bacteria to varying degrees; however, inhibition of GE03231 site accelerated cell autolysis. Therefore, the whole genome crRNA inhibition library is well suited for B. amyloliquefaciens LB1ba02 and can be further applied to high-throughput mining of other functional genes.