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Telomerase represents an excellent target for cancer therapy. Different telomerase activity inhibitors have been developed to treat cancer, but all failed due to side effects. Thanks to CRISPR-Cas9 technology, a cancer gene therapy named telomerase-activating gene expression (Tage) system was developed to target telomerase in cancer cells. The Tage system consists of multiple sequences that are subsequently activated only in those cells with an active telomere synthesis: it all gets triggered by the pairing of the endogenous telomerase to a single stranded sequence upstream of a wild type Cas9, which makes this enzyme elongate this sequence by adding telomeres. Moreover, a single guide RNA that recognizes telomeres and a dead Cas9 (without any nuclease ability) tagged with VP64 are produced under different promoters. The abundance of telomere-targeting sgRNA and dCas9-VP64 allows their pairing, and the complex binds to the telomeres of the formerly described construct. VP64 is a transcriptional activator, and the serial deposition of dCas9-VP64 chimeres upstream of a wild type Cas9 greatly enhances its transcription. As a result, the abundance of wild type Cas9 competes with dCas9-VP64 for the telomere-targeting sgRNA, and this huge amount can sustain the targeting of nucleolar telomeres, cleaving them and killing the cell. Upon some rounds of optimization, the authors effectively induced death in various cancer cells tested (HepG2, HeLa, PANC-1, MDA-MB-453, A549, HT-29, SKOV-3, Hepa1-6) without affecting normal cells (MRC-5, HL7702) and bone marrow mesenchymal stem cell .
Schepers et al. developed a suicide switch system to eliminate the undesirable proliferating cells, allowing the genome-wide CRISPR screening only in growth-arrested subpopulations. Using this system, several autophagy-related proteins were identified as targets for senescence induction in A549 lung cancer cells. The suicide switch system consists of a construct that contains an inducible caspase 9 system (iCasp9) driven by the Ki-67 promoter. The iCasp9 contains the intracellular portion of the human caspase 9 protein, fused to a drug-binding site responsible for the chemical induction of caspase 9 homodimers dimerization. The subsequent addition of the drug AP20187 leads to dimerization of the caspase 9 homodimers, resulting in cellular apoptosis and elimination of the proliferating cells. Then, the growth-arrested subpopulations, identified using specific cytofluorimetric markers, were infected with the Brunello CRISPR library, a library characterized by sgRNAs that target genes known to be associated with senescence. The genome-scale CRISPR screening led to identify ATG9A, RB1CC1, ATG101 and RAB14 as possible targets for senescence-inducing therapies. As a matter of fact, knocking out of these four genes in the proliferating cells induces tumor cell senescence as demonstrated by the expression of the typical senescence markers . 2b1af7f3a8