Efficient CRISPR/Cas9-Mediated Gene Editing in Arabidopsis thaliana and Inheritance of Modified Genes in the T2 and T3 Generations
Figure 5
Efficiency of Cas9/sgRNA mutagenesis in Arabidopsis plants.
PCR/Restriction Enzyme (PCR/RE) analysis of total DNA extracts from individual hygromycin resistant T1 Arabidopsis plants (lanes 1–12) showing the relative proportion of nonfunctional GFP genes mutagenized by Cas9/sgRNA activity. Bottom arrow indicates the expected ∼125 bp DNA fragments resulting from ApaLI cleavage of the ∼250 bp PCR product amplified from nonfunctional, out-of-frame, nonmutagenized GFP genes that contains an intact ApaLI cleavage site in the sgRNA target region. Top arrow indicates the expected ∼250 bp size of PCR products from GFP genes mutagenized by the Cas9/sgRNA system in such a manner that they are no longer are susceptible to cleavage by ApaLI. NG (Nonfunctional GFPGene), the PCR products amplified from a sgRNA target site of a cloned nonfunctional, out-of-frame, GFP gene digested with ApaLI. % modified GFP Gene = (pixels in 250 bp band)/(pixels in 250 bp band+pixels in 125 bp band) ×100.