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The expanding repertoire of programmable nucleases such as Cas9 brings new opportunities in genetic medicine. 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 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. 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. The inhibitor of 53BP1 (or i53) was identified through the screening of a massive combinatorial library of engineered ubiquitin variants by phage display. 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.

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