Fig. 5
From: A RecET-assisted CRISPR–Cas9 genome editing in Corynebacterium glutamicum
Point mutation using the RecET-assisted CRISPR–Cas9 method. a Verification of start codon replacement of pgi gene by sequencing. Plasmids pHAsgRNApgi1 harboring the mutated start codon (ATG to GTG) in HAs and pHAsgRNApgi2 harboring both the mutated start codon in HAs and the mutated nucleotides in PAM site (NGG to NAG) were transformed into EDT by electroporation. Cells were spread on chloramphenicol-resistant BHIS plate for cultivation. PCR amplifications were performed using random-selective colonies as the templates. (n/N): n number of correctly edited transformants, N number of tested transformants. b Mutants verification by sequencing argB gene. To replace the A26V and M31V at the same time, the repair templates were designed to contain 10 mutated base pairs, among which two nucleotides were mutated for amino acid substitution and the other eight nucleotides were mutated to prevent Cas9 cleavage. c The performance of EDT and its mutants for l-arginine production in the shake-flask cultivation. The data are derived from experiments performed at least three times, and the error bars represent the standard deviations