MACBETH: Multiplex automated Corynebacterium glutamicum base editing method

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

“…The plasmids used in this study are listed in Table S3. pnCas9(D10A)‐AID TS and pgRNA‐ ccdB NoPer were used as templates for constructing expression plasmids of base editors (Y. Wang et al, ). Expression of the base editor construct was controlled by an IPTG‐inducible promoter P tac .…”

“…Transcription of the gRNA was controlled by a constitutive promoter P 11F with an unequivocal transcription start site (Figure S2). Application of the promoter P 11F resulted in an extra A at the 5′‐end of the transcribed gRNA but has been proven to be effective for Cas9‐based genome editing in C. glutamicum (J. Liu et al, ; Y. Wang et al, ). nVQR‐Cas9(D10A)‐AID and nVRER‐Cas9(D10A)‐AID expression plasmids were constructed by introducing amino acid substitutions to pnCas9(D10A)‐AID TS through PCR.…”

“…The bioinformatics tool “sgRNAcas9” (Xie, Shen, Zhang, Huang, & Zhang, ) was modified for PAM selection and used to identify all gRNAs of target genes. The Perl algorithm developed in our previous study (Y. Wang et al, ) was then used to screen gRNAs containing editable codons in the editing window (CGA, CAG, CAA, and CCA). The bioinformatics tool “Cas‐OFFinder” (Bae, Park, & Kim, ) was used to search gRNAs’ potential off‐target sites in the reference genome.…”

“…However, the current base editing system requires an NGG protospacer‐adjacent motif (PAM) and have a ~5‐bp editing window. These requirements obviously limit the number of sites in a genome that can be targeted (Y. Wang et al, ). Taking base editing‐mediated gene inactivation as an example, introducing premature stop codons into the first half of target genes is expected to implement gene knockout with confidence.…”

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…Despite the editing efficiency, the availability of editable sites in the target bacterial genome is another restraint of CBE‐mediated gene disruption. Wang et al indicated about 88% genes in C. glutamcium genome (GC content is ≈50%) are available for generating a premature stop codon, regardless of the stop codon position while another evaluation demonstrated that the potential genes for CBE‐mediated disruption were only ≈70% in S. aureus whose GC content is lower than 30% . When the stop codon position was set at the top half of the gene body, the available sites shrank to about 50%.…”