Development of a RecE/T-Assisted CRISPR-Cas9 Toolbox for Lactobacillus

Lactobacilli are members of a large family involved in industrial food fermentation, therapeutics, and health promotion. However, the development of genetic manipulation tools for this genus lags behind its relative industrial and medical significance. The development of clustered regularly interspaced short palindromic repeat (CRISPR)-based genome engineering for Lactobacillus is now underway. However, some Lactobacillus species are sensitive to CRISPR-Cas9 induced double strand breaks (DSBs) due to a deficiency in homology-directed repair (HDR), which allows chromosomal genetic editing. Here, phage-derived RecE/T is coupled with CRISPR-Cas9 and the transcriptional activity of broad-spectrum host promoters is assessed to set up a versatile toolbox containing a recombination helper plasmid and a broad host CRISPR-Cas9 editing plasmid, which enables efficient genome editing in Lactobacillus plantarum (L. plantarum) WCFS1 and Lactobacillus brevis (L. brevis) ATCC367. The RecE/T-assisted CRISPR-Cas9 toolbox realizes single gene deletions at an efficiency of 50-100% in seven days. Furthermore, the chromosomal gene replacement of Lp_0537 using a P₂₃ -pyruvate decarboxylase (pdc) expression cassette is accomplished with an efficiency of 35.7%. This study establises a RecE/T-assisted CRISPR genome editing toolbox for L. plantarum WCFS1 and L. brevis ATCC367 and also demonstrate that RecE/T-assisted CRISPR-Cas9 is an effective genome editing system, which can be readily implemented in Lactobacilli.