Construction of a food-grade gene editing system based on CRISPR-Cas9 and its application in Lactococcus lactis NZ9000

Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system has been widely used in gene editing of various organisms. However, food-grade gene editing systems in lactic acid bacteria are still preliminary. Red/ET-dependent homologous recombination or CRISPR-based systems have been developed to gene editing in Lactococcus lactis, but these methods are overall inefficient. In the present study, a recombinant system based on CRISPR/Cas9 technology combined with Red/ET was developed using the plasmid pMG36e derived from Lactococcus lactis. Then, the developed recombinant system was applied to Lactococcus lactis. Knockout efficiency was significantly higher using the developed system (91%). In addition, this system showed the potential to be used as a high-throughput method for hierarchical screening. Finally, a gene-edited strain was obtained, and no antibiotics or exogenous genes were introduced using the developed gene editing system. Thus, the efficient system in lactic acid bacteria was constructed and optimized.