Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system

• Published in: Microbial cell factories Vol. 16; no. 1; pp. 201 - 13
• Main Authors: Peng, Feng, Wang, Xinyue, Sun, Yang, Dong, Guibin, Yang, Yankun, Liu, Xiuxia, Bai, Zhonghu
• Format: Journal Article
• Language: English
• Published: London BioMed Central 14.11.2017; BioMed Central Ltd; BMC
• Subjects: Amino acids; Analysis; Applied Microbiology; Biotechnology; Chemistry; Chemistry and Materials Science; Chromosome deletion; Chromosomes; Clonal deletion; Cloning; Commodities; Corynebacterium glutamicum; Corynebacterium glutamicum - genetics; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; CRISPR/Cas9 and the microbial cell factory; Deoxyribonucleic acid; Design; DNA; DNA repair; DNA sequencing; E coli; Efficiency; Endonucleases - genetics; Enzymology; Gene Deletion; Gene Editing - methods; Gene expression; Genetic aspects; Genetic Engineering; Genetic modification; Genetic Vectors; Genome editing; Genomes; Gram-positive bacteria; Green fluorescent protein; Homology; Industrial production; Insertion; Investigations; Metabolic pathways; Microbial Genetics and Genomics; Microbiology; Microorganisms; Mutagenesis, Insertional; Mutation; Nucleotide sequence; Plasmids; Point Mutation; Protein expression; Proteins; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems; Signal transduction; CRISPR/Cas9; Protein expression; Genome editing; Corynebacterium glutamicum
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-017-0814-6

Background Corynebacterium glutamicum ( C. glutamicum ) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. Results In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB , mepA , clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB - and mepA -deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. Conclusions In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum . Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.