Low in vivo transgene integration frequency limits the therapeutic efficacy of homology-directed repair (HDR)-mediated gene insertion as a treatment for Mendelian disorders. This study demonstrates improved efficacy of HDR-mediated gene insertion for the treatment of murine phenylalanine hydroxylase (PAH) deficiency, a model of human phenylketonuria (PKU), through pharmacologic inhibition of competing DNA repair pathways. Targeted integration of a Pah -expressing transgene into the hepatocytes of neonatal mice was enhanced with vanillin, a potent inhibitor of non-homologous end joining (NHEJ). This was further improved following combination of vanillin and novobiocin, an inhibitor of microhomology-mediated end joining (MMEJ). Combined NHEJ and MMEJ inhibition yielded PAH-expressing transgene insertions in approximately 10% of targeted alleles and was associated with a 70.6% decrease in serum phenylalanine. Demonstrating that pharmacologic inhibition of DNA repair pathways that compete with HDR can significantly enhance HDR-mediated transgene insertion in vivo .