Fig. 1

Optimization of CRISPR/Cas9 system in C. glutamicum. a Schematic representation of the plasmids used for optimizing the promoter of Cas9 expression. The cas9 ^180bp-rfp fusion gene that consisted of the first 180 bp of cas9 gene and the full-length rfp gene was inserted into pXMJ19 under the control of IPTG-inducible promoter P _tac (pRfp1) or propionate-inducible promoter P _prpD2 (pRfp2). The empty plasmid pXMJ19 was used as a negative control. b Optimization of promoters for Cas9 expression. pRfp1, pRfp2, and pXMJ19 were transformed into C. glutamicum SL4 separately. The resultant transformants were cultivated in SGY medium with or without 1 mM IPTG (for pRfp1) or 1 g/L sodium propionate (for pRfp2). Cells of the stationary growth phase were used to detect their fluorescence outputs using a microplate reader (λ excitation = 560 nm, λ emission = 607 nm). c Schematic representation of the plasmids used for verifying the function of the terminator derived from S. pyogenes (T _Sp ). A rfp gene was inserted downstream the gRNA (pRfp3) and the modified gRNA with T _Sp deleted (pRfp4). The empty plasmid pEC-XK99E was used as a negative control. d Function verification of T _Sp in E. coli and C. glutamicum by detecting fluorescence outputs of strains harboring pRfp3, pRfp4, or pEC-XK99E. e The gRNA structure derived from plasmid pgRNA1. f The optimized gRNA structure derived from plasmid pgRNA2. g Escape rate of CRISPR/Cas9-based counter-selection using different gRNA expression plasmid. pgRNA1 and pgRNA2 were transformed into C. glutamicum SL4 (pCas9) separately. Correct transformants were cultivated, diluted and spread on SGY plates containing Km and Cm, with or without IPTG (1 mM). The escape rate of counter-selection was calculated by colony counting