Improved gene targeting in vivo using EoHR, a small molecule inhibitor of 53BP1
Precise genome editing by programmable nucleases such as Cas9 has revolutionised medical research by enabling the creation of gene-edited or knock-in mouse models of disease. However, a major limitation of the approach is the inefficient process of homology driven recombination (HDR) from an exogenous DNA repair template. This is because error-prone, 53BP1-dependent non-homologous end joining (NHEJ) predominates at Cas9-induced double strand breaks (DSBs). Here we report the validation of a cell-permeable and non-toxic inhibitor of 53BP1 called EoHR. In vitro, EoHR prevents 53BP1 binding to dimethylated lysine 20 on histone H4, a marker of DSBs. In cells, EoHR prevents localisation of 53BP1 at nuclease mediated DSBs and promotes HDR at a Cas9-induced break. When tested in vivo during mouse Cas9-mediated gene editing, inclusion of EoHR at the time of microinjection more than doubled the recovery of correctly edited mice and halved the time to project success. Our work shows that inhibition of 53BP by EoHR is a simple and robust way to increase HDR at Cas9 breaks that can also dramatically increase the success rates of animal model generation.