Extended Data Fig. 1: Generation of isogenic HD403/+ and HD403/403 iPSCs by homology-directed repair.
From: Base editing correction of hypertrophic cardiomyopathy in human cardiomyocytes and humanized mice
a, Using iPSCs derived from a healthy donor (HDWT), the MYH7 p.R403Q (c.1208 G > A) variant was introduced by CRISPR-Cas9-based homology-directed repair (HDR) using SpCas9, a sgRNA (spacer sequence colored in green, PAM sequence colored in gold), and a single-stranded oligodeoxynucleotide (ssODN) donor template containing the PV. A heterozygous genotype (HD403/+) and homozygous genotype (HD403/403) were isolated. Chromatograms highlighting insertion of the PV and corresponding amino acid changes are shown for indicated genotypes. Red arrows indicate coding nucleotide 1208 and amino acid 403. b, Sanger sequencing chromatogram showing no insertion of the PV on the highly homologous MYH6 gene. Red arrow indicates coding nucleotide 1211 and amino acid 404. c, HDWT and HD403/+ iPSCs readily differentiate into CMs. Cardiac troponin I (cTnI, green) highlights CMs; nuclei (blue) are marked by DAPI (4′,6-diamidino-2-phenylindole). Scale bar, 25 μm. Similar ability for iPSCs to differentiate into CMs was found in at least three separate differentiations for each genotype. d, Ratio of MYH7 to MYH6 gene expression in HDWT and HD403/+ iPSC-CMs as measured by quantitative PCR. Data are mean ± s.d. across four separate differentiations.
