Journal
STEM CELL REPORTS
Volume 17, Issue 4, Pages 993-1008Publisher
CELL PRESS
DOI: 10.1016/j.stemcr.2022.02.008
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Funding
- US National Institutes of Health (NIH) National Institute on Neurological Disorders and Stroke (NINDS) [U54NS108874]
- NIH/NINDS [R01NS073873, R56NS073873]
- National Institute on Aging (NIA) [R01NS104219]
- Les Turner ALS Foundation
- New York Stem Cell Foundation
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Editing iPSC cells using CRISPR/Cas9 may result in on-target genomic defects, but there is currently a lack of effective quality control methods. This study describes a cost-efficient strategy that successfully identifies edited clones with detrimental on-target events.
The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.
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