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. 2018 May 8;39(43):3879–3892. doi: 10.1093/eurheartj/ehy249

Figure 1.

Figure 1

CRISPR/Cas9 engineering of MYH7. (A) Schema of MYH7 highlighting target and Protospacer Adjacent Motif (PAM) for nickase CRISPR/Cas9 editing. (B) Genotyping of MYH7 in human pluripotent stem cells, introducing c.C9123T and showing a silent mutation (TGG PAM to TCG). (C) RT-PCR showed selection cassette causes an MYH7 knockout; Flippase-mediated excision restores expression. (D) Immunostaining of cardiac α-actinin human pluripotent stem cell-cardiomyocytes and fibroblast controls (inset); cell mask and DAPI as counterstains. Bar = 100 μm. (E) Cardiomyocyte differentiation purity >90% α-actinin + cells; n = 8; Bar = 100 µm. (F) RFLP of MYH7, with ratiometric densitometry of MUT: wild-type (n = 4). FLP, Flippase; MW, molecular weight; NTC, non-template control. Data, mean ± SD.