Fig. 4

Stimulation of transgene integration by Cas9–HE in iPS cells. a Relative frequencies of HDR induced by Cas9–HE at DNA cleavage site directed by T2 guide RNA. Human iPS cells were transfected with the indicated Cas9 plasmids, T2 guide RNA, and puromycin transgene donor with 800-bp homology arms to the AAVS1 locus. HDR-mediated transgene integration was measured by counting puromycin-resistant clones, resulting from targeted integration at the AAVS1 locus of the puromycin resistance exon carried by the transgene. The results are expressed as the mean relative number of puromycin-resistant clones after normalizing the number of puromycin-resistant clones by that induced by Cas9. The difference between Cas9–HE and Cas9 was statistically significant (t-test, *P<0.05). Data represented are from four independent experiments. Error bars indicate standard deviation. b A clone of puromycin-resistant cells derived from puromycin selection of cells transfected with Cas9–HE, T2 guide RNA, and puromycin-resistant transgene donor was analyzed for expression of stemness markers TRA-1-60 and SSEA4 or Oct3/4 and Sox2 by FACS analysis. c After induction of cardiomyocyte differentiation in the clone analyzed in b, expression of cardiomyocyte markers alpha-actinin and MLC2-v was analyzed by immunohistochemistry. Scale bar, 50 µm