CRISPR-Cas9 Editing of hiPSCs and PHSats

hiPSCs: hiPSCs were harvested as a single-cell suspension and, after washing with DPBS, resuspended in P3 Primary Cell Nucleofector Solution (Lonza, Basel, Switzerland) premixed with NLS-SpCas9-NLS (Aldevron, Fargo, ND) and/or sgRNA specific for CAPN3 c.550delA (Synthego, Menlo Park, CA). For each nucleofection, 300,000 cells with 3 μg SpCas9 and sgRNA in a mass ratio of 1:0.67 was used in a 20 μL reaction (16-well nucleofection cuvette). For mock (unedited), cells were resuspended in P3 Primary Cell Nucleofector Solution. The cells were nucleofected using an Amaxa 4D Nucleofector (Lonza) with the CB-150 program. Afterward, 100 μL mTeSR Plus was added and cells were plated in mTeSR Plus with 10 μM Rock inhibitor Y-27632. The medium was changed next day to mTeSR Plus. PHSats: PHSats were harvested with TrypLE Express and, after washing with DPBS, resuspended in P5 Primary Cell Nucleofector Solution (Lonza) premixed with NLS-SpCas9-NLS (Aldevron) and/or sgRNA specific for CAPN3 c.550delA (Synthego). For each nucleofection 150,000 cells with 3 μg SpCas9 and sgRNA in a mass ratio of 1:0.67 were used in a 20 μL reaction (16-well nucleofection cuvette). For mock (unedited), cells were resuspended in P5 Primary Cell Nucleofector Solution. The cells were nucleofected using an Amaxa 4D Nucleofector (Lonza) with the EY-100 program. Afterward 100 μL SMCGM was added, and cells were plated. The medium was changed next day.