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

Research output: Contribution to journalArticlepeer-review

86 Scopus citations

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
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer Nature America, Inc.

Funding

We thank N. Zelikson (Tel Aviv University) for helpful discussions and for reviewing the manuscript. We thank R. Elkon for computational support. We thank the IDRFU, GRU and SICF units, Tel Aviv University, for logistic support and counsel. This research was funded by a Boaz and Varda Dotan donation (A.B.); H2020 European Research Council grant 759296 570 (A.B.); Israel Science Foundation grants 1632/16, 2157/16 and 2876/21 (A.B.); Israeli Health Ministry grant 0604216191 (A.B.); and National Institutes of Health grant R01 AI167003-01 (A.B.) This research was funded by the European Research Council (759296 to A.B. and 945674 to U.B.-D.); the Gertner Institute Scholarship; the Yoran Institute Scholarship; the SAIA Foundation (A.D.N.); SCGC Tel Aviv University (A.B. and U.B.-D.); the Israel Cancer Research Foundation Development Award (A.M.); the Israel Cancer Research Foundation Gesher Award (U.B.-D.); the Azrieli Faculty Fellowship (U.B.-D.); the Alon Fellowship for Outstanding Young Scientists; the Israel Council for Higher Education (A.M.); and the Edmond J. Safra Center Fellowship for Bioinformatics at Tel Aviv University (E.G.). We thank N. Zelikson (Tel Aviv University) for helpful discussions and for reviewing the manuscript. We thank R. Elkon for computational support. We thank the IDRFU, GRU and SICF units, Tel Aviv University, for logistic support and counsel. This research was funded by a Boaz and Varda Dotan donation (A.B.); H2020 European Research Council grant 759296 570 (A.B.); Israel Science Foundation grants 1632/16, 2157/16 and 2876/21 (A.B.); Israeli Health Ministry grant 0604216191 (A.B.); and National Institutes of Health grant R01 AI167003-01 (A.B.) This research was funded by the European Research Council (759296 to A.B. and 945674 to U.B.-D.); the Gertner Institute Scholarship; the Yoran Institute Scholarship; the SAIA Foundation (A.D.N.); SCGC Tel Aviv University (A.B. and U.B.-D.); the Israel Cancer Research Foundation Development Award (A.M.); the Israel Cancer Research Foundation Gesher Award (U.B.-D.); the Azrieli Faculty Fellowship (U.B.-D.); the Alon Fellowship for Outstanding Young Scientists; the Israel Council for Higher Education (A.M.); and the Edmond J. Safra Center Fellowship for Bioinformatics at Tel Aviv University (E.G.).

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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