Development and application of a CRISPR-dCpf1 assisted multiplex gene regulation system 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, the restriction-modification system, existed in B. amyloliquefaciens LB1ba02, results in a low transformation efficiency, which makes its CRISPR tool development lagging far behind other Bacillus species. Here, we adapted a nuclease-deficient mutant dCpf1 (D917A) of Cpf1 and developed a CRISPR/dCpf1 assisted multiplex gene regulation system for the first time in B. amyloliquefaciens LB1ba02. A 73.9-fold inhibition efficiency and an optimal 1.8-fold activation effect at the - 327 bp site upstream of the TSS were observed in this system. In addition, this system achieved the simultaneous activation of the expression of three genes (secE, secDF, and prsA) by designing a crRNA array. On this basis, we constructed a crRNA activation library for the proteins involved in the Sec pathway, and screened 7 proteins that could promote the secretion of extracellular proteins. Among them, the most significant effect was observed when the expression of molecular motor transporter SecA was activated. Not only that, we constructed crRNA arrays to activate the expression of two or three proteins in combination. The results showed that the secretion efficiency of fluorescent protein GFP was further increased and an optimal 9.8-fold effect was observed when SecA and CsaA were simultaneously activated in shake flask fermentation. Therefore, the CRISPR/dCpf1-ω transcription regulation system can be applied well in a restriction-modification system strain and this system provides another CRISPR-based regulation tool for researchers who are committed to the development of genetic engineering and metabolic circuits in B. amyloliquefaciens.