Journal Article

Frequent aneuploidy in primary human T cells after CRISPR–Cas9 cleavage

Horovitz-Fried, Miriam, Khosravi, Rami, Reinstein, Eyal, Madi, Asaf, Tenne, Tamar, Ben-David, Uri, Reuveni, Eli, Nahmad, Alessio David, Kobo, Hila, Goldschmidt, Ella, Barzel, Adi, Liberman, Meytal

2022

Overview

Multiple clinical trials of allogeneic T cell therapy use site-specific nucleases to disrupt T cell receptor (TCR) and other genes 1 – 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. Aneuploidy and chromosomal truncations are frequent outcomes of CRISPR–Cas9 editing in human T cells.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/61/212/2166 / 692/700/565/251 / Agriculture / Aneuploidy / Apoptosis / Bioinformatics / Biomedical and Life Sciences / Biomedical Engineering/Biotechnology / Biomedicine / Biotechnology / Cell activation / Cell death / Cell therapy / Chromosome 14 / Chromosome 7 / Chromosomes / Cleavage / Clinical trials / CRISPR / CRISPR-Cas Systems - genetics / Editing / Fluorescence in situ hybridization / Gene Editing - methods / Gene sequencing / Genome editing / Genomes / Humans / In Situ Hybridization, Fluorescence / Life Sciences / Lymphocytes / Lymphocytes T / Nuclease / p53 Protein / PD-1 protein / Receptors, Antigen, T-Cell - genetics / T cell receptors / T-Lymphocytes / Transfection