Here we present the discovery and validation of EoHR, the first compound demonstrated in vivo to enhance the type of CRISPR required for specific gene editing. In addition to its prospects for gene therapy, CRISPR is revolutionising medical research by enabling researchers to edit genes to create mouse models of disease and introduce human genes into mice to test therapeutics. However, CRISPR efficiency for large gene insertions into mouse zygotes is poor. This makes obtaining a knock-in mouse a slow, risky and expensive process, usually requiring multiple experimental cycles, each of 6-8 weeks duration. The overall failure rate is disturbingly high, especially when targeting native gene loci. Inserting or specifically editing a gene requires Cas9 endonuclease to create a targeted DNA double-strand break (DSB) followed by homology driven recombination (HDR) of an exogenous DNA repair template. Efficiency is low because non-homologous end joining (NHEJ) is the predominant DSB repair mechanism and is error prone. NHEJ is regulated by the epigenetic reader, 53BP1 binding to dimethylated lysine 20 on histone 4, that signals for nearby DSBs. Here we describe the identification of a compound, EoHR, by fragment-based drug discovery that binds to the dimethylated lysine pocket on the tandem tudor domain of 53BP1. We then show that EoHR prevents the accumulation of 53BP1 at DSBs in real time in live cells by using a 53BP1-GFP construct with an inducible restriction enzyme to create DSBs. In the presence of EoHR the migration of 53BP1 to DSB foci is abolished. We then show that adding EoHR to standard microinjection protocols enhances gene insertion 2.3-fold for the creation of transgenic mice. We do this using the most robust pairwise experimental design, involving 17 independently commissioned projects, each requiring a unique large gene insertion at a distinct locus (n = 797 mice, p = 0.003). Incorporating EoHR into microinjections will accelerate medical research by more than halving the number of cycles required to achieve gene transfer via CRISPR, on average saving months of failed experiments per project. This should substantially reduce the risk and cost for scientists to obtain a knock-in mouse.