Kinetics of Nuclear Uptake and Site-Specific DNA Cleavage during CRISPR-Directed Gene Editing in Solid Tumor Cells

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–directed gene editing is approaching clinical implementation in cancer. Thus, it is imperative to define the molecular framework upon which safe and efficacious therapeutic strategies can be built. Two important reaction parameters include the biological time frame within which the CRISPR/Cas complex enters the nucleus and executes gene editing, and the method of discrimination that the CRISPR/Cas complex utilizes to target tumor cell, but not normal cell, genomes. We are developing CRISPR-directed gene editing for the treatment of non–small cell lung carcinoma focusing on disabling Nuclear Factor Erythroid 2-Related Factor-Like (NRF2), a transcription factor that regulates chemoresistance and whose genetic disruption would enhance chemosensitivity. In this report, we define the time frame of cellular events that surround the initialization of CRISPR-directed gene editing as a function of the nuclear penetration and the execution of NRF2 gene disruption. We also identify a unique protospacer adjacent motif that facilitates site-specific cleavage of the NRF2 gene present only in tumor genomes. Implications: Our results begin to set a scientifically meritorious foundation for the exploitation of CRISPR-directed gene editing as an augmentative therapy for lung cancer and other solid tumors. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/18/6/891/F1.large.jpg.