CRISPRoff Epigenetic Editing Enables Durable Gene Silencing in CAR-T Cell Therapy
Key Insights
Researchers developed CRISPRoff (search), a CRISPR-based epigenetic editing platform that achieves durable gene silencing in primary human T cells without DNA breaks, maintaining silencing through approximately 30-80 cell divisions over 28 days.
The technology enables multiplexed targeting of up to five genes simultaneously with minimal cellular toxicity, contrasting with nuclease-active Cas9 (search) which causes substantial toxicity when targeting multiple genes.
Integration with CAR-T cell manufacturing demonstrated enhanced therapeutic efficacy, with RASA2 (search)-silenced CAR-T cells showing significantly better tumor control and extended survival in preclinical leukemia (search) models compared to control cells.
A breakthrough in epigenetic editing technology has demonstrated the ability to achieve durable gene silencing in primary human T cells without causing DNA breaks, potentially transforming approaches to cellular immunotherapy. Researchers from the University of California, San Francisco and Arc Institute (search) have developed CRISPRoff (search), a CRISPR-based platform that programs stable gene silencing through DNA methylation, offering significant advantages over traditional nuclease-based approaches.
The study, published in Nature Biotechnology, establishes CRISPRoff (search) as a powerful tool for enhancing CAR-T cell therapy (search) by enabling simultaneous genetic and epigenetic modifications without the cytotoxicity associated with multiple DNA breaks.
Durable Silencing Across Cell Divisions
The research team compared CRISPRoff (search) with CRISPRi (search) and Cas9 (search) nuclease across multiple endogenous gene targets over 28 days. While CRISPRi-mediated silencing was progressively lost over time, particularly upon T cell restimulation, CRISPRoff programmed durable gene silencing comparable to Cas9 knockout. The silencing persisted through three rounds of anti-CD2/CD3/CD28 antibody restimulation, demonstrating stable propagation across approximately 30 to 80 cell divisions in vitro.
RNA sequencing confirmed highly specific targeting, with robust repression of intended genes and no off-target differentially expressed genes at 28 days after electroporation. Whole-genome bisulfite sequencing revealed that the highest differentially methylated region between targeting and control samples occurred precisely at the targeted locus.
The platform proved effective across diverse genomic contexts. For genes with CpG islands, including therapeutically relevant targets such as FAS (search), PTPN2 (search), RC3H1 (search), SUV39H1 (search), MED12 (search) and RASA2 (search), at least one guide RNA achieved potent and durable silencing. The team also successfully targeted genes lacking CpG island annotations, achieving stable silencing of CD5 (search) and LAG3 (search) that remained 99.5% and 99.1% silenced, respectively, at 30 days, whereas PDCD1 (search) showed 78% silencing in bulk populations.
Multiplexed Targeting Without Toxicity
A critical advantage emerged when targeting multiple genes simultaneously. Targeting three, four or five genes with nuclease-active Cas9 (search) resulted in substantial cellular toxicity, likely due to multiple DNA breaks. In contrast, CRISPRoff (search) multiplexed targeting produced minimal toxicity while achieving combined silencing of three, four, and five target genes at 93.5%, 82.4%, and 65.8% efficiency, respectively, across four donors at 30 days.
"CRISPRoff (search) 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 (search)-mediated genome editing," the researchers note in the manuscript.
Enhanced CAR-T Cell Function
The clinical potential of this technology was validated through integration with CAR-T cell manufacturing. The researchers developed orthogonal CRISPR systems using Acidaminococcus species Cas12a ribonucleoproteins for targeted CAR knock-in at the TRAC (search) locus, combined with CRISPRoff (search)-mediated silencing of RASA2 (search), a gene whose ablation promotes T cell function across immunosuppressive conditions.
This combined approach maintained efficient CAR integration whilst achieving robust gene silencing. In repetitive stimulation assays, RASA2 (search)-silenced CAR-T cells continued to kill target cells efficiently after five rounds of stimulation. In contrast, control-edited cells progressively declined in their ability to control cancer (search) cells.
The enhanced functionality translated to improved outcomes in preclinical models. In NSG mice bearing Nalm6 leukaemia, animals treated with RASA2 (search)-silenced CAR-T cells showed significantly better tumour control and extended survival compared to control CAR-T cells across multiple independent experiments and four donors.
Stable In Vivo Performance
Importantly, CRISPRoff (search)-induced silencing remained stable following in vivo adoptive transfer. When CD151 (search)-silenced CAR-T cells were transferred into mice bearing subcutaneous A375 melanoma (search) tumours, highly efficient knockdown was maintained in CAR-T cells isolated from tumours and spleens 14 days after transfer, confirming that epigenetic silencing persists through tumour antigen recognition and the in vivo environment.
"Epi-edited CAR-T cells can maintain stable target gene silencing even through multiple rounds of successful antigen-positive cancer (search) cell killing, enabling functional enhancement of CAR-T cells through silencing of 'checkpoint' genes without the need for multiplexed gene cleavage," the authors emphasise.
Expanding Epigenetic Capabilities
The researchers also demonstrated CRISPRon (search), a complementary technology for targeted gene activation through DNA demethylation. By targeting the FOXP3 Treg-specific demethylated region (TSDR) in conventional CD4+ T cells, they achieved stable upregulation of FOXP3 expression over 28 days, suggesting potential applications for generating regulatory T cells for autoimmune diseases.
The study establishes a comprehensive epigenetic editing toolkit for primary human T cells, offering new possibilities for cellular immunotherapy enhancement without the safety concerns associated with multiple DNA breaks. The technology's ability to maintain stable modifications through extensive cell divisions and in vivo conditions positions it as a promising platform for next-generation cell therapies.
Laine Goudy and Luke Gilbert led the study from the University of California, San Francisco and Arc Institute (search). The research represents a significant advancement in the field of epigenetic editing, providing a safer alternative to nuclease-based approaches for complex genetic modifications in therapeutic cells.