Development of Base Editors for Simultaneously Editing Multiple Loci in Lactococcus lactis

Lactococcus lactis serves as the most extensively studied model organism and an important dairy species. Though CRISPR-Cas9 systems have been developed for robust genetic manipulations, simultaneously editing multiple endogenous loci in L. lactis is still challenging. Herein, we first report the development of a double-strand break-free, robust, multiloci editing system CRISPR-deaminase-assisted base editor (CRISPR-DBE), which comprises a cytidine (CRISPR-cDBE) and an adenosine deaminaseassisted base editor (CRISPR-aDBE). Specifically targeted by a sgRNA, CRISPR-cDBE can efficiently introduce a cytidine-tothymidine mutation and CRISPR-aDBE can high-efficiently convert adenosine to guanosine within a 5 nt editing window. CRISPRcDBE was validated and successfully applied to simultaneously inactivate multiple genes using a single plasmid in L. lactis strain NZ9000. Meanwhile, the temperature-sensitive plasmid of CRISPR-DBE can be cured quickly, and the continuous gene editing of L. lactis has been achieved. Furthermore, CRISPR-cDBE can also efficiently convert the targeted C to T in a nisin-producing, industrial L. lactis strain F44. Finally, we applied genome-wide bioinformatics analysis to determine the scope of gene inactivation for these base editors using different Cas9 variants and evaluated the preference of SpGn and SpRYn variants for the protospacer adjacent motif in L. lactis NZ9000. Taken together, our study provides a powerful tool for simultaneously editing multiple loci in L. lactis, which may have a wide range of industrial applications in the future.

Automated summary

This study reports the development of a CRISPR-based multi-loci editing system that can efficiently edit multiple loci in L. lactis, providing a powerful tool for industrial applications.