Park, Jaehyun SHIN HYOJUNG Lee, Sun-Mi Um, Youngsoon Woo, Han Min 2024-01-19T23:34:44Z 2024-01-19T23:34:44Z 2021-09-03 2018-01 1475-2859 https://pubs.kist.re.kr/handle/201004/121894 Background: The construction of microbial cell factories requires cost-effective and rapid strain development through metabolic engineering. Recently, RNA-guided CRISPR technologies have been developed for metabolic engineering of industrially-relevant host. Results: To demonstrate the application of the CRISPR interference (CRISPRi), we developed two-plasmid CRISPRi vectors and applied the CRISPRi in Corynebacterium glutamicum to repress single target genes and double target genes simultaneously. Four-different single genes (the pyc, gltA, idsA, and glgC genes) repressions were successfully performed using the CRISPRi vectors, resulting significant mRNA reductions of the targets compared to a control. Subsequently, the phenotypes for the target gene-repressed strains were analyzed, showing the expected cell growth behaviors with different carbon sources. In addition, double gene repression (the idsA and glgC genes in a different order) by the CRISPRi resulted in an independent gene repression to each target gene simultaneously. To demonstrate an industrial application of the CRISPRi, citrate synthase (CS)-targeting DM1919 (L-lysine producer) strains with a sgRNA-gltA-r showed reduced CS activity, resulting in the improvement of L-lysine yield by 1.39-fold than the parental DM1919 (a lysine producer). Conclusions: Single or double gene repression were successfully performed using the CRISPRi vectors and sequence specific sgRNAs. The CRISPRi can be applied for multiplex metabolic engineering to enhanced lysine production and it will promote the further rapid development of microbial cell factories of C. glutamicum. English BioMed Central RNA-guided single/double gene repressions in Corynebacterium glutamicum using an efficient CRISPR interference and its application to industrial strain Article 10.1186/s12934-017-0843-1 1 Microbial Cell Factories, v.17 Microbial Cell Factories 17 Y scie scopus 000419616200003 2-s2.0-85040323809 Biotechnology & Applied Microbiology Biotechnology & Applied Microbiology Article SEQUENCE-SPECIFIC CONTROL LYSINE PRODUCTION ESCHERICHIA-COLI CITRATE SYNTHASE EXPRESSION PLATFORM ACETATE GLUCOSE GROWTH Metabolic engineering Synthetic biology CRISPR interference Corynebacterium glutamicum