Functional characterization of tumor-specific CRISPR-directed gene editing as a combinatorial therapy for the treatment of solid tumors.

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.