Editing stem cell genomes at scale to measure variant effects in diverse cell and genetic contexts.

Multiplexed assays of variant effect (MAVEs) systematically measure variant function but have been limited to cancer cell lines rather than disease-relevant cell types. We developed saturation genome editing in human iPSCs (iPSC-SGE) to introduce variant libraries into a single allele of a target gene while programming the genetic background of the second allele, enabling variant assessment across differentiated cell types and genetic contexts at scale. We edited 1,137 variants into MYBPC3 and measured protein abundance in cardiomyocytes and cardiac organoids, accurately identifying pathogenic variants, and resolving variants of uncertain significance. Highlighting the importance of genetic context, we edited 437 POLG variants in two genetic backgrounds and identified loss-of-function and dominant-negative variants. Finally, we illuminate a path for scaling iPSC-SGE by identifying 443 disease genes essential for iPSC or iPSC-derived neuron growth. iPSC-SGE enables systematic assessment of variants in specialized human cell types, advancing MAVEs to empower genomic medicine.