2.1. Cell Culture and Reagents

BJFF.6 iPSCs (Cellosaurus, CVCL-VU02 [40]) and AN1.1 iPSCs (Washington University, St. Louis, MO, USA; T-019532) were cultured in StemFlex media (Gibco, Carlsbad, CA,
USA), supplemented with 1% (v/v) antibiotic/antimycotic (Gibco) and 5% (v/v) CO2 in a humidified incubator at 37 ?C. Edited iPSC pools were generated using CRISPR-Cas9
technology and homologous DNA donors. One million iPSCs were nucleofected (4D Nucleofector ™ X-unit; Lonza, Basel, Switzerland) per the manufacturer’s recommended
protocol with pre-complexed ribonucleoprotein (RNPs) consisting of 100 pmol chemically modified single guide RNA (sgRNA; Integrated DNA Technologies, Coralville, IA, USA) (Supplementary Table S1) and 33 pmol 3XNLS SpCas9 protein (St. Jude Children’s Research Hospital [SJCRH] Protein Production Facility, Memphis, TN, USA) using P3 nucleofector solution and CA-137 program in 20-µL or 100-µL cuvettes. Control cell pools contained a non-targeting sgRNA and a non-targeting DNA donor to control for RNP, and donor concentrations added to an HDRescue editing experiment. Unless otherwise noted, editing rates were assessed on day 10 post-transfection.

U2OS cells (ATCC, HTB-96) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Corning, 10-013-CV, Corning, NY, USA) supplemented with 10% (v/v) FBEssence,
1% (v/v) penicillin-streptomycin, and 1X GlutaMAX at 37 ?C, 5% v/v CO2 in a humidified incubator. U2OS cell pools were generated by nucleofecting 500,000 cells as described above using P3 nucleofector solution (Lonza) and the CM-104 program in 20-µL cuvettes. Unless otherwise noted, editing rates were assessed on day 28 post-transfection. 

HeLa cells (ATCC, CRM-CCL-2) were cultured in DMEM supplemented with 10% (v/v) FBEssence, 1% (v/v) penicillin-streptomycin, and 1X GlutaMAX at 37 ?C,
5% v/v CO2 in a humidified incubator. HeLa cells pools were generated by nucleofecting 500,000 HeLa cells as described above using SE nucleofector solution (Lonza) and DS-150 program in 20-µL cuvettes. Unless otherwise noted, editing rates were assessed on day 16 post-transfection.