Abstract N-WASP and WASP can induce actin polymerization via Arp2/3 and were reported to be crucial for homology-directed repair (HDR) of DNA double-strand breaks (DSB). The underlying mechanism was suggested to involve nuclear actin polymerization, but the mechanistic details were debated. Unexpectedly, we show now that neither WASP nor N-WASP is required for HDR during CRISPR-mediated genome editing. Using knock-out and overexpression of N-WASP and WASP in U2OS cells, we did not detect alterations in total gene editing, HDR, or the ratio of HDR to non-homologous end joining (NHEJ) as assessed by different methods. Furthermore, we could not observe colocalization of HA-tagged WASP or N-WASP with DSBs. Finally, while the Arp2/3 inhibitor CK-666 and ARPC4 knockdown by siRNA reduced HDR efficiency in U2OS cells, this corresponded with a decreased transfection efficiency and a reduction of the HDR-proficient cell cycle phases S and G2/M. In summary, contrary to expectations, these data do not support a crucial role for N-WASP and WASP in DSB repair.
Phan et al. (Fri,) studied this question.