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The integration of the lentiviral DNA into the host genome is completed by cellular factors that repair the short gaps flanking the proviral DNA. Several repair complexes have been implicated to participate in the repair of the integration intermediate including components of the non-homologous DNA repair pathway (NHEJ) [1]. NHEJ is generally initiated by the binding of Ku heterodimer to a double strand DNA break. However, no DSBs are formed during lentiviral integration. We have shown earlier, that HIV-1 integrase can directly interact with Ku70 repair factor, and this interaction is weakened by E212A/L213A substitutions in integrase [2]. Overexpression of an integrase-binding domain of Ku70 in cells reduces the transduction by a single round replication incompetent CMV-driven HIV-1 vector but has no influence on the vector bearing the E212A/L213A substitutions. This effect may be caused by an inhibition of the interaction between integrase and the endogenous Ku70 protein by an integrase-interacting Ku70 domain. Using CRISPR/Cas9 technology we have established a set of 293T derived sublines with a stable depletion of either Ku70, Ku80 or DNA-PKcs subunits of the DSB detection complex. The depletion of any subunit resulted in a decrease in HIV-1 single cycle replication which be caused by a compromised integration or by a reduction in the postintegrational gap repair. To choose between these two possibilities, we have established a qPCR-based approach for a quantitative measurement of postintegrational gap repair. We have shown that depletion of any of the DNA-PK components reduced gap repair efficiency in our system. The same effect has been detected in the presence of specific DNA-PKcs inhibitor Nu7441 on 293T cells as well as on the lymphoid Jurkat cells. Viral vector carrying E212A/L213A integrase substitutions demonstrates decreased gap repair and a reduced sensitivity to the DNA-PK components depletion while its integrational capacity remains at the wild type level. We speculate that integrase recruits DNA-PK complex to gaps and facilitates gap repair through a direct interaction with Ku70 subunit.