Fig. 1.

"i-CRISPR" strategy kills cancer cells by inducing DSBs in corresponding mutation sites. A Diagram of our proposed personalized CRISPR-mediated cancer treatment strategy named "i-CRISPR". The basic mechanism of radiotherapy is to induce DNA damage, especially DSBs, through radiation [5, 6]. When accumulated DNA breaks, especially DSBs, cannot be repaired, death signals are often activated. If DSBs are generated specifically in cancer cells through personalized CRISPR scissors and the repair of these DSBs is intensively blocked, specific killing of cancer cells may be achieved. Although the basic cell-killing mechanisms of our strategy and radiotherapy are similar, our strategy is more precise and personalized. Because both DNA damage repair inhibitors (i) and CRISPR are necessary, so we named this strategy "i-CRISPR". B Representative images of γH2AX foci in HepG2 cells at 48 h after transfection with three groups of gRNAs together with Cas9. To block the repair of DSBs, gene-edited cells were also treated with the ATM inhibitor KU55933 (10 μM), the DNA-PKcs inhibitor NU7441 (10 μM), or the combination of KU55933 (10 μM) + NU7441 (10 μM). Y: Quantitative analysis of the γH2AX foci number in the different groups indicated above. *P < 0.05. **P < 0.01. C, D At 0, 24, 48, and 72 h after gRNA transfection, HepG2 and Hep3B cells were pretreated with DMSO, KU55933, NU7441 and KU55933 + NU7441, and cell viability was determined with a CCK-8 assay at OD 450. E Representative images of cell apoptosis determined by flow cytometry analysis in cells transfected with three groups of gRNAs and Cas9 and treated with different inhibitors. F, G Quantitative analysis of cell apoptosis (Annexin V positive) and necrosis (PI positive, Annexin V negative) at 48 h after transfection combined with DNA repair inhibitor treatments. H Representative images of organoids (HCC-227) transfected with Cas9 and/or gRNAs combined with DNA damage repair inhibitor treatment. And the average number of organoids per field were quantified. I Tumor volume were recorded every three days after the injection of gRNA and DSB inhibitor. And tumor growth curve was obtained from the indicated two groups. *P < 0.05