Fig. 1: Gene deletion, insertion, and ssDNA recombineering in C. glutamicum using the optimized CRISPR/Cas9 system.

a Deletion of a 1.7 kb DNA fragment (cgl0620-cgl0622) using the all-in-one plasmid. Different concentrations of IPTG (0.01, 0.05, and 0.5 mM) were used for counter-selection. b Deletion of the same 1.7 kb DNA fragment (cgl0620-cgl0622) using the optimized all-in-one plasmid with a modified lac operator that binds LacI very tightly (LacO^*) and a weak RBS (RBS2). Different concentrations of IPTG (0.01, 0.05, and 0.5 mM) were used for counter-selection. c Deletion and insertion of large DNA fragments. Three tests, deletion of a 20 kb DNA fragment (part of prophage CGP3) with 0.5 kb HR arms, deletion of a 219 kb DNA fragment (complete prophage CGP3) with 1.0 kb HR arms, and insertion of a 4 kb DNA fragment (an artificial proBAC operon inserted to putA) with 1.0 kb HR arms were conducted. For gene insertion, the inserted DNA fragment and HR arms were provided using a second plasmid. IPTG (0.05 mM) was used for counter-selection. For a, b, and c, twenty-three colonies were randomly selected and verified by PCR for each test. Results of three independent replicates are shown. Red and grey fractions of bars represent edited and unedited colonies, respectively. The average editing efficiency (%) from three independent replicates is shown above the bars. d Introduction of triple and single nucleotide changes using CRISPR/Cas9-assisted ssDNA recombineering. Three tests, triple nucleotide changes with 90 nt ssDNA (two independent replicates), single nucleotide change with 90 nt ssDNA, and triple nucleotide changes with 60 nt ssDNA, were conducted to introduce rpsL^K43R mutation that produced streptomycin resistance phenotype. IPTG (0.05 mM) was used for counter-selection. Thirty colonies were randomly selected and verified by streptomycin resistance phenotype test. Red and grey fractions of bars represent edited and unedited colonies, respectively. The editing efficiency (%) is shown above the bars. Genetic elements on the plasmids: P_tac, IPTG-inducible tac promoter; P_11F, 11F constitutive promoter³²; P_ddh^*, a variant of the promoter of ddh gene (Supplementary Data 6); LacO, wild-type lac operator; LacO^*, a modified lac operator that binds LacI tightly⁴²; RBS1, a strong RBS AAAGGAGTTGAGA; RBS2, a weak RBS AAAGGCACCCGAT; pUC ori, pUC origin of replication; pBL1 ori, pBL1 origin of replication; pGA1 ori, pGA1 origin of replication; cas9, S. pyogenes Cas9 gene; gRNA, guide RNA expression cassette; HR arms, homologous recombination arms; cm^R, chloramphenicol resistance gene; km^R, kanamycin resistance gene; recT, gene encoding the recombinase from the Rac prophage of E. coli; Insertion, the 4 kb DNA fragment for insertion test. Images for DNA gel electrophoresis and streptomycin resistance phenotype test are provided in Supplementary Figs. 1 and 3. Source data underlying Fig. 1 are provided as a Source Data file.