Repurposing endogenous type II CRISPR‐Cas9 system for genome editing in Streptococcus thermophilus

Streptococcus thermophilus has been extensively used in industrial milk fermentation. However, lack of efficient genetic manipulation approaches greatly hampered the industrial application of this species. Here, we repurposed the endogenous CRISPR1 and CRISPR3 systems, both belong to type II-A CRISPR-Cas9, by delivering a self-targeting CRISPR array with DNA repair template into S. thermophilus LMD-9. We achieved 785-bp deletion in lacZ gene by repurposing CRISPR1 and CRISPR3 systems with efficiencies of 35% and 59%, respectively, when 1-kb DNA repair template was provided. While providing with 1.5-kb repair template, the editing efficiency for deletion in lacZ gene reached 90% using CRISPR3 systems. Diverse editing outcomes encompassing a stop code insertion and single nucleotide variation within lacZ, as well as a 234-bp DNA fragment insertion upstream of ster_0903, were generated with high efficiencies of 75%-100% using the CRISPR3 system. Harnessing the customized endogenous CRISPR3 system to target six genes of eps gene cluster, we obtained six single-gene knockout mutants with efficiencies of 29%-80%, and proved that the epsA, epsE, and epsG were the key genes affecting exopolysaccharides biosynthesis in S. thermophilus LMD-9. Altogether, repurposing the native type II-A CRISPR-Cas9 can be served as a toolkit for precise genome engineering in S. thermophilus for biotechnological applications.