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Updated: Sep 6, 2025
CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions
Published on: November 1, 2024
Frequent aneuploidy in primary human T cells after CRISPR-Cas9 cleavage
Alessio David Nahmad1,2, Eli Reuveni3, Ella Goldschmidt4
1School of Neurobiology, Biochemistry and Biophysics, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Abstract:
Multiple clinical trials of allogeneic T cell therapy use site-specific nucleases to disrupt T cell receptor (TCR) and other genes1-6. In this study, using single-cell RNA sequencing, we investigated genome editing outcomes in primary human T cells transfected with CRISPR-Cas9 and guide RNAs targeting genes for TCR chains and programmed cell death protein 1. Four days after transfection, we found a loss of chromosome 14, harboring the TCRα locus, in up to 9% of the cells and a chromosome 14 gain in up to 1.4% of the cells. Chromosome 7, harboring the TCRβ locus, was truncated in 9.9% of the cells. Aberrations were validated using fluorescence in situ hybridization and digital droplet PCR. Aneuploidy was associated with reduced proliferation, induced p53 activation and cell death. However, at 11 days after transfection, 0.9% of T cells still had a chromosome 14 loss. Aneuploidy and chromosomal truncations are, thus, frequent outcomes of CRISPR-Cas9 cleavage that should be monitored and minimized in clinical protocols.
Insights
CRISPR-Cas9 gene editing in T cells can cause significant chromosomal abnormalities, including loss and truncation. These unintended genomic alterations impact cell function and survival, requiring careful monitoring in clinical applications.
Area of Science:
- Genomics
- Immunotherapy
- Cell Biology
Background:
- Allogeneic T cell therapy utilizes gene editing to modify T cells for therapeutic purposes.
- Site-specific nucleases, such as CRISPR-Cas9, are employed to disrupt genes like the T cell receptor (TCR).
- Understanding the precise genomic outcomes of CRISPR-Cas9 editing in primary human T cells is crucial for clinical safety.
Purpose of the Study:
- To investigate the genome editing outcomes in primary human T cells following CRISPR-Cas9 transfection.
- To assess the frequency and nature of chromosomal aberrations induced by CRISPR-Cas9 targeting TCR genes and programmed cell death protein 1.
- To evaluate the impact of these aberrations on T cell viability and function.
Main Methods:
- Primary human T cells were transfected with CRISPR-Cas9 and guide RNAs targeting TCR and programmed cell death protein 1 genes.
- Single-cell RNA sequencing was used to analyze genome editing outcomes.
- Fluorescence in situ hybridization and digital droplet PCR were employed for aberration validation.
- Cell proliferation, p53 activation, and cell death were assessed.
Main Results:
- CRISPR-Cas9 editing resulted in significant chromosomal abnormalities, including loss of chromosome 14 (up to 9%) and truncation of chromosome 7 (up to 9.9%).
- Aneuploidy, such as chromosome 14 gain (up to 1.4%), was also observed.
- These chromosomal aberrations were associated with reduced T cell proliferation, p53 activation, and increased cell death.
- Chromosomal abnormalities persisted, with chromosome 14 loss detected in 0.9% of cells at 11 days post-transfection.
Conclusions:
- CRISPR-Cas9 cleavage frequently leads to aneuploidy and chromosomal truncations in human T cells.
- These genomic alterations can negatively affect T cell function and survival.
- Clinical protocols using CRISPR-Cas9 for T cell therapy must incorporate monitoring and mitigation strategies for these unintended genomic consequences.
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