CRISPR-to-Kill (C2K)–Employing the Bacterial Immune System to Kill Cancer Cells

SIMPLE SUMMARY: Reasoning that multiple DNA breaks will trigger programmed cell death, we generated lentiviral CRISPR-to-kill (C2K) vectors targeting highly repetitive SINE sequences for cancer gene therapy. In proof-of-concept experiments, C2K-Alu-vectors selectively killed human, but not murine cell lines, and efficiently inhibited the growth of patient-derived glioblastoma cell lines resistant to high-dose irradiation. In combination with tumor-targeting approaches, the C2K system might represent a promising tool for cancer gene therapy. ABSTRACT: CRISPR/Cas9 was described as a bacterial immune system that uses targeted introduction of DNA double-strand breaks (DSBs) to destroy invaders. We hypothesized that we can analogously employ CRISPR/Cas9 nucleases to kill cancer cells by inducing maximal numbers of DSBs in their genome and thus triggering programmed cell death. To do so, we generated CRISPR-to-kill (C2K) lentiviral particles targeting highly repetitive Short Interspersed Nuclear Element-Alu sequences. Our Alu-specific sgRNA has more than 15,000 perfectly matched target sites within the human genome. C2K-Alu-vectors selectively killed human, but not murine cell lines. More importantly, they efficiently inhibited the growth of cancer cells including patient-derived glioblastoma cell lines resistant to high-dose irradiation. Our data provide proof-of-concept for the potential of C2K as a novel treatment strategy overcoming common resistance mechanisms. In combination with tumor-targeting approaches, the C2K system might therefore represent a promising tool for cancer gene therapy.