CRISPR/Cas9 was described as a bacterial “immune system” that uses targeted introduction of DNA double-strand breaks (DSBs) to destroy nucleic acids of foreign origin. In the majority of CRISPR/Cas9 medical applications, the goal is to make it as precise and specific as possible. Additional DSBs might be a source of unpredictable side effects caused by faulty repair leading to, e.g., gene knockouts, chromosomal aberrations, or exhaustion of repair capabilities resulting in cell-cycle arrest and programmed cell death (PCD). We hypothesized that we could take advantage of the latter process to deliberately trigger PCD in malignant cells. The human and other eukaryotic genomes contain noncoding repetitive elements of several types. These repetitive elements can be used to introduce large numbers of DSBs into the human genome with one single guide RNA (sgRNA). 

To this aim, we generated CRISPR-to-kill (C2K) lentiviral and virus-like particles (LV and VLP) targeting highly repetitive Short Interspersed Nuclear Elements SINE/Alu sequences to trigger programmed cell death. The selected Alu-specific sgRNA has over 15,000 perfectly matched target sites within the human genome. We demonstrate that C2K-Alu-vectors selectively killed human, but not murine cell lines. More importantly, they efficiently inhibited the growth of cancer cells, including largely treatment-resistant patient-derived, “stem-like” glioblastoma cell lines. Our data provide proof-of-concept for the potential of C2K as a novel treatment strategy hard to be overcome by common resistance mechanisms. In combination with tumour-targeting approaches, the C2K system might therefore represent a promising tool for cancer gene therapy.