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 language | English |
|---|---|
| Pages (from-to) | 1807-1813 |
| Number of pages | 7 |
| Journal | Nature Biotechnology |
| Volume | 40 |
| Issue number | 12 |
| DOIs | |
| State | Published - Dec 2022 |
| Externally published | Yes |
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.).
| Funders | Funder number |
|---|---|
| Alon Fellowship for Outstanding Young Scientists | |
| Varda and Boaz 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 Diseases | R01AI167003 |
| Israel Cancer Research Fund | |
| Georgia Regents University | |
| H2020 European Research Council | 759296 570 |
| European Commission | 945674, 759296 |
| Israel Science Foundation | 2157/16, 1632/16, 2876/21 |
| Tel Aviv University | |
| Council for Higher Education | |
| Ministry of Health, State of Israel | 0604216191 |