Development of Base Editors for Simultaneously Editing Multiple Loci in .

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 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 deaminase-assisted base editor (CRISPR-aDBE). Specifically targeted by a sgRNA, CRISPR-cDBE can efficiently introduce a cytidine-to-thymidine mutation and CRISPR-aDBE can high-efficiently convert adenosine to guanosine within a 5 nt editing window. CRISPR-cDBE was validated and successfully applied to simultaneously inactivate multiple genes using a single plasmid in strain NZ9000. Meanwhile, the temperature-sensitive plasmid of CRISPR-DBE can be cured quickly, and the continuous gene editing of has been achieved. Furthermore, CRISPR-cDBE can also efficiently convert the targeted C to T in a nisin-producing, industrial 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 NZ9000. Taken together, our study provides a powerful tool for simultaneously editing multiple loci in , which may have a wide range of industrial applications in the future.