Integrated epigenetic and genetic programming of primary human T cells
Targeted epigenetic engineering of gene expression in cell therapies would allow programming of desirable phenotypes without many of the challenges and safety risks associated with double-strand break-based genetic editing approaches. Here, we develop an all-RNA platform for efficient, durable and multiplexed epigenetic programming in primary human T cells, stably turning endogenous genes off or on using CRISPRoff and CRISPRon epigenetic editors. We achieve epigenetic programming of diverse targeted genomic elements without the need for sustained expression of CRISPR systems. CRISPRoff-mediated gene silencing is maintained through numerous cell divisions, T cell stimulations and in vivo adoptive transfer, avoiding cytotoxicity or chromosomal abnormalities inherent to multiplexed Cas9-mediated genome editing. Lastly, we successfully combined genetic and epigenetic engineering using orthogonal CRISPR Cas12a-dCas9 systems for targeted chimeric antigen receptor (CAR) knock-in and CRISPRoff silencing of therapeutically relevant genes to improve preclinical CAR-T cell-mediated in vivo tumor control and survival.
Authors
- Nupura Kale (ORCID: https://orcid.org/0000-0002-3185-8646)
- Philip J. Santangelo (ORCID: https://orcid.org/0000-0001-7352-0339)
- Chang Liu
- Joseph J. Muldoon (ORCID: https://orcid.org/0000-0003-3784-3565)
- Laine Goudy
- Ling Chow
- Jennifer M. Umhoefer (ORCID: https://orcid.org/0000-0002-8563-9641)
- Julia Carnevale (ORCID: https://orcid.org/0000-0001-9410-7148)
- Luke A. Gilbert (ORCID: https://orcid.org/0000-0001-5854-0825)
- Justin Eyquem (ORCID: https://orcid.org/0000-0001-8262-1190)
- Rosmely Hernandez (ORCID: https://orcid.org/0000-0002-2998-2437)
- Jae Hyun J. Lee
- Brian R. Shy (ORCID: https://orcid.org/0000-0001-9569-3708)
- Alvin Ha (ORCID: https://orcid.org/0000-0002-7092-9072)
- Alexis Talbot (ORCID: https://orcid.org/0000-0001-6734-413X)
- Ashir A. Borah (ORCID: https://orcid.org/0000-0003-1193-4987)
- Alexander Marson (ORCID: https://orcid.org/0000-0002-2734-5776)
- Zhongmei Li (ORCID: https://orcid.org/0000-0001-8616-9568)
- Abolfazl Arab (ORCID: https://orcid.org/0000-0002-0024-2937)
- Ralf Schmidt (ORCID: https://orcid.org/0000-0002-6697-0363)
- Sandesh Subramanya (ORCID: https://orcid.org/0000-0002-2503-7200)
- C C Tran
Institutions
- Georgia Institute of Technology (US)
- Gladstone Institutes (US)
- Inserm (FR)
- University of California, San Francisco (US)
- Université Paris Cité (FR)
- American Research Center in Egypt (US)
- The Wallace H. Coulter Department of Biomedical Engineering (US)
- Innovative Genomics Institute (US)
- Parker Institute for Cancer Immunotherapy (US)
- Palo Alto Institute (US)
- Hôpital Saint-Louis (FR)
- UCSF Helen Diller Family Comprehensive Cancer Center (US)
- Arc Research Institute
- Medical University of Vienna (AT)
Publication Details
- Journal
- Nature Biotechnology
- Published
- 2025-10-21
- DOI
- https://doi.org/10.1038/s41587-025-02856-w
- Citations
- 12
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 9.67
Funders
- National Science Foundation
- Cancer Research Institute
- Parker Institute for Cancer Immunotherapy
- Emerson Collective
- National Institutes of Health
- University of California, San Francisco
- National Cancer Institute