TY - JOUR
T1 - Frequent aneuploidy in primary human T cells after CRISPR–Cas9 cleavage
AU - Nahmad, Alessio David
AU - Reuveni, Eli
AU - Goldschmidt, Ella
AU - Tenne, Tamar
AU - Liberman, Meytal
AU - Horovitz-Fried, Miriam
AU - Khosravi, Rami
AU - Kobo, Hila
AU - Reinstein, Eyal
AU - Madi, Asaf
AU - Ben-David, Uri
AU - Barzel, Adi
N1 - Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer Nature America, Inc.
PY - 2022/12
Y1 - 2022/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85133192220&partnerID=8YFLogxK
U2 - 10.1038/s41587-022-01377-0
DO - 10.1038/s41587-022-01377-0
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C2 - 35773341
AN - SCOPUS:85133192220
SN - 1087-0156
VL - 40
SP - 1807
EP - 1813
JO - Nature Biotechnology
JF - Nature Biotechnology
IS - 12
ER -
