Fig. 3

Measuring KIT copy number reduction in porcine kidney cells transfected with CRISPR/Cas9. a Schematic illustration of the Nla III assay. Red arrows indicate the first nucleotide in intron 17. The G > A mutation introduces an Nla III restriction site, as labelled by the blue underline. The bases with a yellow background represent exon 17 and those with a grey background represent intron 17. Blue arrowheads indicate the cutting sites of Nla III. b Variation of the percentage of KIT copy with the splice mutation in cell populations transfected with CRISPR/Cas9 as determined by the Nla III assay. M, DNA ladder; D, duroc pig (pig breed with wild-type KIT allele); NC, negative control, denotes the unedited Yorkshire pig kidney cells; US, unsorted; S, sorted. Red arrowheads indicate the expected positions of DNA bands cleaved by Nla III. The numbers along the bottom of the gel indicate the A/(G + A) ratio calculated based on the band intensities using Image J software. c The A/(G + A) ratio in cell populations transfected with CRISPR/Cas9 as measured by TA clone sequencing analysis. d KIT copy number variations in cell populations transfected with CRISPR/Cas9 as determined by qPCR (T-test, p < 0.05)