Figure 2

In vitro detection of CRISPR-mediated mutations. To confirm the activity of CRISPR-mediated targeted gene mutation, chicken fibroblasts were seeded in 96-well plates. Varying concentrations of CRISPR::pp38 plasmid DNA (0.2, 0.4, 0.6, 0.8 and 1 μg/well) were transfected using a lipofection procedure. The cells were incubated in complete DMEM for 36 h. Genomic DNA was isolated, and the target pp38 gene was amplified by PCR. The gel-purified DNA fragment was digested with the restriction enzyme PspF1. Uncut DNA was extracted, cloned into the TA vector, and sequenced to detect the frameshift mutation.