Single-plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis

Lactococcus lactis is a food-grade lactic acid bacterial species that widely used in food and medical industries. Due to its relatively small genome simple metabolism, L. commonly engineered produce large quantities of recombinant proteins. The most common single-gene knockout strategy involves RecA-dependent homologous double-crossover recombination, which time-consuming laborious. In this study, precise efficient genome-editing plasmid for NZ9000 engineering, pLL, was established based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. By studying the effects different single guide RNA (sgRNA) promoters, efficiency gene deletion optimized. For LLNZ_02045 (ldh), up 50% achieved. Effective sequential LLNZ_11240 (upp) LLNZ_04580 (upp1) also demonstrated using tool. Additionally, encodes uracil phosphoribosyltransferase identified system. Similar robust efficiencies sgRNA targeted regions suggested not affected by location binding. Thus, our study new gene-editing tool may allow further investigation understanding genome.