| Title: |
Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls |
| Authors: |
Simkin, Dina; Papakis, Vasileios; Bustos, Bernabe I; Ambrosi, Christina M; Ryan, Steven J; Baru, Valeriya; Williams, Luis A; Dempsey, Graham T; McManus, Owen B; Landers, John E; Lubbe, Steven J; George, Alfred L; Kiskinis, Evangelos |
| Contributors: |
Neurology |
| Source: |
Stem cell reports ; 17 ; 4 ; 993 ; 1008 ; United States |
| Publication Year: |
2023 |
| Collection: |
University of Massachusetts, Medical School: eScholarship@UMMS |
| Subject Terms: |
CRISPR/Cas9; KCNQ2; WGS; disease modeling; genome editing; iPSCs; isogenic control lines; mtDNA; on-target insertions/deletions |
| Description: |
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. |
| Document Type: |
article in journal/newspaper |
| File Description: |
application/pdf |
| Language: |
English |
| Relation: |
Stem Cell Reports; https://doi.org/10.1016/j.stemcr.2022.02.008; http://hdl.handle.net/20.500.14038/52437 |
| DOI: |
10.1016/j.stemcr.2022.02.008 |
| Availability: |
https://doi.org/10.1016/j.stemcr.2022.02.008; https://hdl.handle.net/20.500.14038/52437 |
| Rights: |
Copyright 2022 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ; Attribution-NonCommercial-NoDerivatives 4.0 International ; http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| Accession Number: |
edsbas.466CEF38 |
| Database: |
BASE |