[An efficient marker-free genome editing method for ].

is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of . is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for . , but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for . based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in ) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for . .