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

摘要

Lactococcus lactis is a food-grade lactic acid bacterialspecies that is widely used in food and medicalindustries. Due to its relatively small genome andsimple metabolism, L. lactis is commonly engineered toproduce large quantities of recombinant proteins. Themost common single-gene knockout strategy in L. lactisinvolves RecA-dependent homologous double-crossoverrecombination, which is relatively time-consuming andlaborious. In this study, a precise and efficient genomeeditingplasmid for L. lactis NZ9000 genome engineering,pLL, was established based on clustered regularlyinterspaced short palindromic repeats (CRISPR)-Cas9technology. By studying the effects of different singleguide RNA (sgRNA) promoters, the efficiency of genedeletion was optimized. For LLNZ_02045 (ldh), genedeletion efficiency of up to 50% was achieved. Effectivesequential gene deletion of LLNZ_11240 (upp)and LLNZ_04580 (upp1) was also demonstrated usingthis tool. Additionally, the gene that encodes for uracilphosphoribosyltransferase was identified using this system.Similar robust gene deletion efficiencies of sgRNAthat targeted different regions of a single gene suggestedthat gene deletion was not affected by the locationof sgRNA binding. Thus, our study established a newgene-editing tool that may allow further investigationand understanding of the L. lactis NZ9000 genome.