可编辑核酸酶介导的哺乳动物基因敲入技术最新进展

Latest Progress of Gene Knock-in Mediated by Programmable Nucleases in Mammals

实验哺乳动物模型是研究基础生物学及人类疾病的重要工具,对实现转基因操作,尤其是基因敲入(knock-in,KI),具有重大意义。锌指核酸酶(zinc-fi nger nucleases,ZFN)、类转录激活因子效应物核酸酶(transcription activator-like effector nucleases,TALEN)和RNA介导的、基于成簇的规律间隔的短回文重复序列和Cas9蛋白的DNA核酸内切酶[clustered regulatory interspaced short palindromic repeat(CRISPR)/Cas9-based RNA-guided DNA endonucleases,CRISPR/Cas9]等的出现,为科研工作者提供了革命性的转基因操作工具。这些可编辑核酸酶通过在靶标序列位置产生双链断裂缺口(double strand breaks,DSBs),并在同源修复模板存在的情况下发生同源重组,进而实现精确的基因敲入。该文主要综述了这些技术的原理及其在哺乳动物KI中取得的最新进展、提高KI效率以及降低脱靶效应的举措等,将有助于KI技术在未来转基因实践中的广泛应用。

Mammalian model animals have contributed significantly to the exploration of basic biology and human diseases. The feasibility to achieve genome engineering in mammals, especially targeted gene knock- in （KI） is critical in this course. Zinc-finger nucleases （ZFN）, transcription activator-like effector nucleases （TALEN） and clustered regulatory interspaced short palindromic repeat （CRISPR）/Cas9-based RNA-guided DNA endonucleases （CRISPR/Cas9） are sparking a new revolution in biological research. These programmable nucleases enable relatively efficient and precise KI by inducing targeted DNA double-strand breaks （DSBs） that stimulate homologous recombination （HR） at the presence of homologous repair templates. Here, we review the principles of these technologies and their applications for gene KI in mammals. We also discuss latest strategies to elevate KI efficiency and reduce off-target ratio, which will enhance the overall performance of future transgenic practice.