The control hiPSC line was kindly provided by Dr. Jianyi Zhang (University of Alabama at Birmingham), this hiPSC line was reprogrammed from cardiac fibroblasts of a healthy female donor and modified to overexpress cyclin D2 to enhance the yield of cardiac differentiation (Zhu et al., 2018 (link); Zhao et al., 2021 (link)). To generate mutant hiPSCs, 2 h prior to electroporation, hiPSCs were treated with 10 μM ROCK inhibitor (Y27632 dihydrochloride, Tocris). Cells were then passaged and 250 K cells were then spun down in PBS. Cells were resuspended in P3 Primary Cell Nucleofector™ Solution containing Supplement 1 (Lonza, Switzerland) and 1 μg of chemically modified sgRNA (Synthego, Menlo Park, CA) and 1.5 μg codon optimized BE4 mRNA (TriLink Biotechnologies, San Diego, CA) and placed in the 20 μL cuvette provided and CB-150 was the Amaxa protocol used. Cells were given 10 μM ROCKi for 48 h post electroporation followed by normal culturing conditions. For hiPSC single cell isolation ∼300 single cell suspended iPSC were cultured in a 10 cm dish with 10 μM ROCKi for 48 h. For the next 10 days cells were given daily media changes and then colonies were isolated using a 10 μL pipette to scrape half of the colony for isolated culture and half the colony for genomic DNA isolation.
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