Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency

Marella D Canny, Nathalie Moatti, Leo C K Wan, Amélie Fradet-Turcotte, Danielle Krasner, Pedro A Mateos-Gomez, Michal Zimmermann, Alexandre Orthwein, Yu-Chi Juang, Wei Zhang, Sylvie M Noordermeer, Eduardo Seclen, Marcus D Wilson, Andrew Vorobyov, Meagan Munro, Andreas Ernst, Timothy F Ng, Tiffany Cho, Paula M Cannon, Sachdev S SidhuFrank Sicheri, Daniel Durocher

Research output: Contribution to journalLetterpeer-review

Abstract

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

Original languageEnglish
Pages (from-to)95-102
Number of pages8
JournalNature Biotechnology
Volume36
Issue number1
DOIs
Publication statusPublished - 27 Nov 2017

Keywords

  • Animals
  • CRISPR-Cas Systems/genetics
  • DNA Damage/genetics
  • DNA End-Joining Repair/genetics
  • DNA Repair/genetics
  • Gene Editing
  • Gene Expression Regulation/genetics
  • Humans
  • Mice
  • Recombinational DNA Repair/genetics
  • Tumor Suppressor p53-Binding Protein 1/antagonists & inhibitors

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