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Updated: Jan 9, 2026
Production of Human CRISPR-Engineered CAR-T Cells
Published on: March 15, 2021
Functional characterization of tumor-specific CRISPR-directed gene editing as a combinatorial therapy for the
Kelly H Banas1, Pawel A Bialk1, Natalia Rivera-Torres1
1Gene Editing Institute, ChristianaCare Health System, Newark, DE 19713, USA.
Abstract:
As we pursue clinical applications for CRISPR-directed gene editing in overcoming resistance to anticancer drugs, we have focused on genetic disruption of the transcription factor, NRF2, a master regulator of cellular stress and detoxification. The level of NRF2 in tumor cells is often a clear determinant of the effectiveness of standard of care. We began to craft a therapeutic approach using tumor-specific CRISPR editing, building upon our previous work elucidating the effect of NRF2 knockout. We selected a prevalent mutation, R34G, that occurs in the Neh2 domain of NRF2, which has been shown to disrupt KEAP1-mediated degradation, thus impacting the NRF2-KEAP1 pathway. Here, we take a global approach by assessing the genomic, transcriptomic, proteomic, and phenotypic profile of a CRISPR-targeted population of cells, both in vitro and in vivo. We detail the design and generation of a clinically relevant cell model and its translation into an animal model, characterizing the efficacy of disabling NRF2 concomitant with the restoration of chemosensitivity. We demonstrate that 20%-40% gene editing activity is sufficient to improve response to chemotherapy in animal models. We suggest that understanding the genetic diversity of CRISPR outcomes must be a key consideration in identifying effective CRISPR molecules for clinical application.
Insights
CRISPR gene editing can overcome cancer drug resistance by disrupting the NRF2 pathway. Even partial disruption of NRF2 in tumor cells restores chemotherapy sensitivity, offering a promising therapeutic strategy.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Editing Technologies
Background:
- NRF2 is a key regulator of cellular stress and detoxification, influencing cancer drug resistance.
- Elevated NRF2 levels in tumor cells often predict poor response to standard chemotherapy.
- The NRF2-KEAP1 pathway is frequently dysregulated in cancer, particularly via mutations like R34G in the Neh2 domain.
Purpose of the Study:
- To investigate the therapeutic potential of CRISPR-directed gene editing to disrupt NRF2 for overcoming anticancer drug resistance.
- To assess the global genomic, transcriptomic, proteomic, and phenotypic effects of CRISPR-targeted NRF2 disruption.
- To evaluate the efficacy of NRF2 knockout in restoring chemosensitivity in preclinical cancer models.
Main Methods:
- Development of a clinically relevant cell model with CRISPR-targeted NRF2 disruption.
- In vitro and in vivo assessment of NRF2 knockout effects.
- Analysis of genomic, transcriptomic, proteomic, and phenotypic profiles.
- Evaluation of chemosensitivity restoration in animal models.
Main Results:
- CRISPR editing targeting the R34G NRF2 mutation was successfully generated in a cell model.
- Disruption of NRF2, even at 20%-40% gene editing activity, significantly improved response to chemotherapy in vivo.
- Comprehensive profiling confirmed the phenotypic and molecular consequences of NRF2 disruption.
Conclusions:
- Targeting NRF2 via CRISPR gene editing is a viable strategy to enhance chemotherapy efficacy and overcome drug resistance.
- Partial gene editing efficiency is sufficient for therapeutic benefit, suggesting feasibility for clinical translation.
- Understanding the genetic diversity of CRISPR outcomes is crucial for selecting optimal CRISPR tools for therapeutic applications.
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