Development of an efficient iterative genome editing method in Bacillus subtilis using the CRISPR‐AsCpf1 system

Bacillus subtilis is a useful chassis in the fields of synthetic biology and metabolic engineering for chemical production. Here, we constructed CRISPR‐AsCpf1‐based expression plasmids with the temperature‐sensitive replicon for iterative genome editing in B. subtilis. This method allowed gene insertion and large genomic deletion with an editing efficiency of up 80%–100% and rapid plasmid curing to facilitate the iterative genome editing in B. subtilis 168. Using the customized CRISPR‐AsCpf1 system, we successfully and efficiently implemented the related gene editing in B. subtilis 168 for hyaluronic acid (HA) biosynthesis, HA synthase gene (hasA) insertion, UDP‐glucose‐dehydrogenase gene (tuaD) insertion, and eps gene cluster (epsA‐O) deletion. The heterologous production of HA was realized by the engineered strain with a yield of 1.39 g/L. These results support the finding that the CRISPR‐AsCpf1 system is highly efficient in bacteria genome editing and provide valuable guidance and essential references for genome engineering in B. subtilis using the CRISPR‐AsCpf1 system.

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