Fig. 1

Application of the CRISPR interferences in C. glutamicum. a Scheme of the CRISPRi system that requires co-expressing a catalytically inactive version of Cas9 (dCas9) protein and a programmable single guide RNA (sgRNA) for the gene of interest (GOI). dCas9 recognizes the PAM sequence (5′-NGG-3′). A programmable sgRNA with dCas9 was designed to block the binding of RNA polymerase. b Two-plasmid system of the CRISPRi for C. glutamicum: pCoryne-dCas9 expresses dCas9 under the tetA promoter and pCoryne-sgRNA expresses a single sgRNA (base-paring region, dCas9 handle, and S. pyogenes terminator) under a constitutive promoter. c Application of the CRISPRi (dCas9-sgRNA complex) to either the wild-type or l-lysine producer (DM1919) by repressing mRNA expression of the chromosomal pyc gene or gltA gene. d Sequences of the PAM sites (blue) and protospacers (red) for CRISPRi of the pyc or gltA genes. The -35 and -10 regions in the promoter DNA sequence are shown in a box. Transcriptional start sites are shown with black arrows. The start codons for translation are underlined. Specific sgRNA names are shown next to the protospacer. The plasmids containing sgRNAs are listed in Table 1