Fig. 4

ssDNA-directed recombineering using CRISPR/Cas9 and RecT in C. glutamicum SL4ΔldhA::rfp. a Schematic representation of pCas9 and pgRNA5 which were used for ssDNA-directed recombineering. b Operation scheme of ssDNA-directed recombineering using CRISPR/Cas9 and RecT for rfp deactivation. A 90mer ssDNA targeted to the lagging strand (rfp-off1, Additional file 1: Table S1) is designed to introduce two successive stop codons (highlighted in red) in rfp gene. PAM sequence of the gRNA is shaded grey. Plasmid pgRNA5 was first transformed into C. glutamicum SL4ΔldhA::rfp. The resultant strain SL4ΔldhA::rfp (pgRNA5) was cultivated in SGY medium supplemented with Km and sodium propionate to induce RecT expression. Electrocompetent cells were then prepared and transformed with pCas9 and ssDNA. After recovery, cells were spread on SGY plates supplemented with Km, Cm, and IPTG (1 mM) to induce Cas9 expression for counter-selection. Colonies were picked randomly and verified by measuring their fluorescence outputs. c Fluorescence output detection of candidate mutants of C. glutamicum SL4ΔldhA::rfp ^off1. –, wild-type C. glutamicum SL4 control; +, C. glutamicum SL4ΔldhA::rfp; 1–10, ten colonies. This displayed the result of Replicate 1. The results of Replicate 2 and Replicate 3 were shown in Additional file 1: Figure S6. d rfp gene sequencing of C. glutamicum SL4ΔldhA::rfp ^off1 mutants. Nucleotides in red box represents the two successive stop codons (TAG TAA) introduced by ssDNA-directed recombineering. e Str^R phenotype test of C. glutamicum SL4 rpsL ^K43R mutants. Ten colonies were picked randomly from SGY plates supplemented with Km, Cm, and IPTG (1 mM) and patched onto SGY plates (Str⁻) and SGY plates supplemented with streptomycin (Str⁺). Seven out of ten colonies were Str^R. f rpsL gene sequencing of C. glutamicum SL4 rpsL ^K43R mutants. Nucleotides in red box represents the mutated codon (AAG to CGC) introduced by ssDNA-directed recombineering