Gene-editing technology can artificially modify genetic material of targeted loci by precise insertion, deletion, or replacement in the genomic DNA. In recent years, with the developments of zinc-finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) technologies, such precise modifications of the animal genomes have become possible. Although gene-editing tools, such as CRISPR/Cas9, can efficiently generate double-strand breaks (DSBs) in mammalian cells, the homology-directed repair (HDR) mediated knock-in (KI) efficiency is extremely low. In this review, we briefly describe the current development of gene-editing tools and summarize the recent strategies to enhance the CRISPR/Cas9- mediated KI efficiency, which will provide a reference for the generation of human disease models, research on gene therapy and livestock genetic improvement.
基因编辑技术是指通过人为方式在基因组插入、缺失或替换特定碱基,对遗传物质进行精确修饰和定向编辑的一种技术。近年来,锌指核酸内切酶(zinc-finger endonuclease, ZFN)、类转录激活因子效应物核酸酶(transcription activator-like effector nuclease, TALEN)、成簇规律间隔短回文重复序列及其相关系统(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9, CRISPR/Cas9)等基因编辑技术的出现,使特异性靶向修饰动物基因组序列成为可能。虽然利用CRISPR/Cas9等基因编辑工具可以在细胞基因组高效产生双链断裂(double-strand breaks, DSB),但利用同源定向修复(homology directed repair, HDR)介导的精确插入(knock in, KI)效率却十分低下。本文结合当前基因编辑技术的发展现状,对目前提高CRISPR/Cas9介导的动物基因组KI策略进行了综述,以期为人类疾病模型制备、基因治疗和家畜遗传改良等提供借鉴。.
Keywords: CRISPR/Cas9; gene editing; homology directed repair; knock in; non-homologous end joining.