A genetically encoded inhibitor of 53BP1 to stimulate homology-based gene editing

The expanding repertoire of programmable nucleases such as Cas9 brings new opportunities in genetic medicine 1–3 . In many cases, these nucleases are engineered to induce a DNA double-strand break (DSB) to stimulate precise genome editing by homologous recombination (HR). However, HR efficiency is nearly always hindered by competing DSB repair pathways such as non-homologous end-joining (NHEJ). HR is also profoundly suppressed in non-replicating cells, thus precluding the use of homology-based genome engineering in a wide variety 4 of cell types. Here, we report the development of a genetically encoded inhibitor of 53BP1 (known as TP53BP1), a regulator of DSB repair pathway choice 5 . 53BP1 promotes NHEJ over HR by suppressing end resection, the formation of 3-prime single-stranded DNA tails, which is the rate-limiting step in HR initiation. 53BP1 also blocks the recruitment of the HR factor BRCA1 to DSB sites in G1 cells 4,6 . The inhibitor of 53BP1 (or i53) was identified through the screening of a massive combinatorial library of engineered ubiquitin variants by phage display 7 . i53 binds and occludes the ligand binding site of the 53BP1 Tudor domain with high affinity and selectivity, blocking its ability to accumulate at sites of DNA damage. i53 is a potent selective inhibitor of 53BP1 and enhances gene targeting and chromosomal gene conversion, two HR-dependent reactions. Finally, i53 can also activate HR in G1 cells when combined with the activation of end-resection and KEAP1 inhibition. We conclude that 53BP1 inhibition is a robust tool to enhance precise genome editing by canonical HR pathways.