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. 2021 Apr 2;22(7):3704. doi: 10.3390/ijms22073704

Figure 1.

Figure 1

Integration of the Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins)9 cassette at the pyrE locus. (A) Schematic representation of the process of integrating the Cas9 cassette at the pyrE locus using the pINT_Cas9 plasmid in Clostridium acetobutylicum. Cas9 in combination with the guide RNA (gRNA) caused a double-strand break at the intergenic region, selecting only cells that have undergone a spontaneous homologous recombination event, resulting in the insertion of the Cas9 cassette. (B) PCR amplification using primers PS1 and PS2 showing the correct integration of the Cas9 cassette at the pyrE locus, amplification results in a 2425 bp band for the wild type, and an 8250 bp band when the Cas9 cassette was integrated. Lane M, 2-log DNA ladder (NEB); H20, water control; Δ1502, MGCΔcac1502 gDNA; 1–4, CAS1 (Δcac1502, pyrE::cas9) clones 1–4.