Frequent Aneuploidy in Primary Human T Cells after CRISPR-Cas9 cleavage

Multiple clinical trials of allogeneic T cell therapy use site-specific nucleases to disrupt T cell receptor (TCR) and other genes (1–6). Here, 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 post-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 post-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.