The CRISPR journalOct 1, 2026

Unexpected Results of CRISPR-Cas9 Gene Editing Found by Detailed DNA Analysis in Identical Human Stem Cell Clones

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Oct 2, 2026

Abstract

Hybridization capture-based targeted next-generation sequencing identified complex structural rearrangements in edited human iPSCs that conventional methods missed.

CRISPR-Cas9 genome editing may cause unintended mutations that complicate understanding of genotype-phenotype relationships.
In human iPSCs with a tandem duplication of the STAG2 locus, hybridization capture-based sequencing revealed inversions and plasmid DNA integrations.
Conventional PCR-based genotyping failed to detect these complex on-target alterations, leading to potential misinterpretations.
Targeted sequencing allowed for a reassessment of editing outcomes, aligning genotypic and transcriptomic data.
The findings suggest hybridization capture-based sequencing could be a practical quality-control method for edited iPSC clones.

Simplified

Advances in CRISPR-Cas9-based genome editing have simplified the generation of isogenic induced pluripotent stem cell (iPSC) pairs differing by a single genetic perturbation, minimizing interindividual variability in disease modeling. However, imprecise editing can cause unintended mutations that distort genotype-phenotype interpretation. Here, we investigated the molecular consequences of CRISPR-Cas9 editing in human iPSCs carrying a tandem duplication of the STAG2 locus. Using hybridization capture-based targeted next-generation sequencing of genomic regions surrounding Cas9-induced double-strand breaks, we identified inversions, plasmid DNA integrations, and complex structural rearrangements that had been missed by conventional PCR-based genotyping. The main advance of our study is the application of a practical hybridization capture-based workflow for resolving such hidden editing outcomes during the generation of isogenic iPSC models. In the selected clones subjected to detailed analysis, targeted sequencing enabled us to revise the initial interpretation of editing outcomes and resolve discrepancies between genotypic and transcriptomic data. Our results demonstrate that complex on-target alterations can remain undetected by conventional PCR-based genotyping and show that hybridization capture-based targeted sequencing can serve as a practical quality-control approach for the characterization of edited iPSC clones.