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

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)1807-1813
Number of pages7
JournalNature Biotechnology
Volume40
Issue number12
DOIs
StatePublished - Dec 2022

Funding

FundersFunder number
Alon Fellowship for Outstanding Young Scientists
Boaz and Varda Dotan donation
Edmond J. Safra Center
IDRFU
SAIA Foundation
SCGC Tel Aviv University
Yoran Institute
National Institutes of Health
National Institute of Allergy and Infectious DiseasesR01AI167003
Israel Cancer Research Fund
Georgia Regents University
H2020 European Research Council759296 570
European Research Council945674, 759296
Israel Science Foundation2157/16, 1632/16, 2876/21
Tel Aviv University
Council for Higher Education
Ministry of Health, State of Israel0604216191

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