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

Corresponding author: Daniel Durocher

Programmable nucleases (可设计的核酸酶), such as Cas9, are used for precise (精确的) genome editing by homology-dependent repair (HDR; 同源依赖性修复). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair (双链断裂修复) pathways, including non-homologous end-joining (NHEJ; 非同源末端连接). 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 (切除), 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 (基因靶向) and chromosomal gene conversion (基因转换) with either double-stranded DNA or single-stranded oligonucleotide donors (寡核苷酸供体) by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

可进行设计的核酸酶，比如Cas9用来通过同源依赖性修复HDR进行精确的基因组编辑。然而包括非同源末端连接NHEJ在内的其它的双链断裂DSB修复通路会与HDR竞争，导致HDR的效率是受到限制。本文报道了在人类和小鼠细胞中通过遗传编码53BP1的抑制因子可以增加HDR依赖型的基因组编辑。53BP1是真核细胞中DSB修复通路选择的关键调控子，主要通过抑制HDR起始中限速阶段的末端切除来使得DNA修复偏向于NHEJ。作者筛选了一个已经构建的针对53BP1抑制因子的泛素变种UbV库。在人类和小鼠细胞中表达53BP1抑制因子会阻断DNA损伤位点上53BP1的积累，提高双链DNA或单链寡核苷酸供体的基因靶向和染色体基因转换约5.6倍。本文显示53BP1的抑制是提高基于HDR的精确基因编辑效率的有效方法。

通讯：Daniel Durocher (http://www.lunenfeld.ca/researchers/durocher)

个人简介：蒙特利尔大学，学士；麦吉尔大学，博士；英国剑桥大学，博士后。

doi: 10.1038/nbt.4021