Figure 5
From: A simple method using CRISPR-Cas9 to knock-out genes in murine cancerous cell lines
Multiple selection markers did not increase the efficiency to obtain KO clones. (A, B) The schemas of knockout of the target gene by pX330 plasmids, ssODNs, and the cassette with PuroR and BSD as selection markers (A) or with HygR, PuroR, and BSD as selection markers (B). The concentration of antibiotics for selection is 100 µg/mL BSD and 5 µg/mL Puromycin (A) or 100 µg/mL BSD, 5 µg/mL Puromycin, and 400 µg/mL Hygromycin (B). Agarose gel images show the genotyping results. The table indicates the genotyping results of 93 clones. BSD, blasticidin S resistant gene; HygR, hygromycin resistant gene; PuroR, puromycin resistant gene; ssODNs, single-strand oligodeoxynucleotides. 5′, 5′ site between the genomic region and the cassette; 3′, 3′ site between the genomic region and the cassette; WT, wild-type allele; −/−, homozygously knockout clones: −/−*, homozygously knockout clones without the detection of 5′ or 3′ sites ligation between the genomic region and the cassette; %KI, percentage of knock-in in 93 clones. (C) Bar graph of KO percentage comparing different approaches in this study.
