Fig. 3

Stimulation of transgene integration by Cas9–HE and Cas9–Geminin. Relative frequencies of HDR and indels induced by Cas9 or fusion of Cas9 to HE domain, Geminin degron, or both. Human HEK293 cells were transfected with the indicated Cas9 plasmids, T2 guide RNA, and GFP transgene donor with homology arms to the AAVS1 targeted locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells, resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequency calculated by normalizing every HDR or indel frequency by that induced by Cas9. Asterisks indicate that the difference is statistically significant when comparing Cas9–HE, Cas9–HE–Geminin, and Cas9–Geminin to Cas9 in t-test (*P<0.05 or **P<0.005). Data are from three independent experiments. Error bars indicate standard deviation. Relative expression levels of Cas9 and other fusions were analyzed by western blot with anti-Cas9 and control anti-tubulin antibodies. Protein extracts were obtained with lysis buffer containing 150 mM NaCl, which resulted in inefficient solubilization of Cas9 fusions with the HE domain compared to those of Cas9 and Cas9–Geminin