Base editing technology based on clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) is a recent addition to the family of CRISPR technologies. Compared with the traditional CRISPR/Cas9 technology, it does not rely on DNA double strand break and homologous recombination, and can realize gene inactivation and point mutation more quickly and simply. Herein, we first developed a base editing method for genome editing in utilizing CRISPR/dCas9 (a fully nuclease-deficient mutant of Cas9 from ) and activation-induced cytidine deaminase (AID). This method achieved three and four loci simultaneous editing with editing efficiency up to 100% and 50%, respectively. Our base editing system in has a 5 nt editing window, which is similar to previously reported base editing in other microorganisms. We demonstrated that the plasmid curing rate is almost 100%, which is advantageous for multiple rounds of genome engineering in . Finally, we applied multiplex genome editing to generate a 168 mutant strain with eight inactive extracellular protease genes in just two rounds of base editing and plasmid curing, suggesting that it is a powerful tool for gene manipulation in and industrial applications in the future.
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