A CRISPR/Cas9-based screening for non-homologous end joining inhibitors reveals ouabain and penfluridol as radiosensitizers

Non-homologous end joining (NHEJ) is the major pathway responsible for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), and correspondingly regulates the cellular response to IR. Identification of NHEJ inhibitors could substantially enhance the tumor radiosensitivity and improve the therapeutic efficiency of radiotherapy. In present study, we demonstrated a screening for NHEJ inhibitors by using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and high-resolution melting (HRM) analysis. Since NHEJ is regarded as an error-prone mechanism, the NHEJ-mediated ligation of the site-specific DSB induced by Cas9 nuclease would eventually cause the mutation of the targeted sequence. Then, HRM analysis, a reliable and rapid assay for detecting sequence variation, was performed to evaluate the mutation efficiency of the targeted site. Validating analysis confirmed the NHEJ activities was positively correlated with the mutation frequencies. Next, an approved drug library containing 1540 compounds was interrogated by using this screening strategy. Our results identified ouabain, a cardiotonic agent, and penfluridol, an antipsychotic agent, have the capacity to restrain NHEJ activity. Further experiments in vitro revealed the radiosensitizing effects of these compounds. Overall, we presented a cell-based screening for NHEJ inhibitors which could promote the discovery of novel radiosensitizers.

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