Generating isogenic hiPSC lines with MYH7 variants

Patient-derived hiPSCs corresponding with non-variant MYH7 (WT Ib) and heterozygous MYH7^E848G/+ variant were previously generated [6 (link)]. 1000k pelleted hiPSCs were mixed with 1 μL 10 μM SP-dCas9-VPR (Addgene, 63798), 9 μL Buffer R2 (STEMCELL Technologies, 100-0691), 1 μL 30 μM of gRNA (Table 2), and 1.5 μg of pJet-MYH7-EGFP-PGK-PuroR plasmid (Supp. File 1). For TP53 ablation, pJet plasmid was omitted. Cells were electroporated at 1400 V for 20 ms with a 10 μL tip using the Neon Transfection System (Thermo Fisher, MPK5000). Transfected cells were plated in mTeSR+ without penicillin/streptomycin supplemented with CloneR2 (STEMCELL Technologies, 100-0691) and 10 μM ROCK inhibitor on plates coated with 80 μg/mL Matrigel. Cells were fed every other day with mTeSR+ with 0.175 μg/mL puromycin dihydrochloride (Thermo Fisher, A1113803) and replated at 88 cells/cm² in a 10 cm plate coated with 80 μg/mL Matrigel for colony picking. Clones were replated in 96 well plates for expansion and genomic DNA harvesting.