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PreprintCited by 4CRISPR and Genetic EngineeringGenomics and Rare DiseasesRNA regulation and disease

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

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Fayer, Shawn, Garge, Riddhiman K., Hopkins, Melissa, Friedman, Clayton E., McGee, Abby V., Rico, Joshua, Powell, Rachel L., McDermot, Evan +13 more·2025·medRxiv
DOI 10.1101/2025.11.12.25340127PMC PMC12642741
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Abstract

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.

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Cited by

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Authors
Fayer, S., Garge, R. K., Hopkins, M., Friedman, C. E., McGee, A. V., Rico, J., Powell, R. L., McDermot, E., Smith, N. T., Pendyala, S., Richardson, M. E., Smith, E. D., Bowen, B. M., Resnick, R., Gupta, P., Stergachis, A. B., Gifford, C., Pinglay, S., Yang, K.-C., Fowler, D. M., & Starita, L. M.
Affiliations
  • University of Washington (US)
  • Stanford Medicine (US)
  • Ambry Genetics (United States) (US)
Corresponding author
Fayer, Shawn
ORCID
Venue
medRxiv
Publisher
openRxiv
Published
2025-11-14
DOI
10.1101/2025.11.12.25340127
PMC
PMC12642741
OpenAlex
W4416347536
Cited by
4
References
4
Citations by year
2026: 4
Type
posted content
Language
en
Open access
Licence
Locations
  • medRxiv (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by)
Funders
  • National Human Genome Research Institute (RM1HG010461, R01HG013025, T32HG000035)
  • National Institute of Diabetes and Digestive and Kidney Diseases (P30DK017047)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Keywords
saturation genome editing, MAVE, iPSC-derived cardiomyocytes, cardiac organoids, MYBPC3, DNA polymerase gamma, variants of uncertain significance, loss-of-function mutations, dominant-negative mutant, iPSC-derived neurons, genetic background, cardiomyopathy

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