《生命科学》 2018, 30(9): 967-979
摘 要：摘 要：基因组靶向敲入是指将特定的外源核酸序列转入细胞或个体基因组中的特定位点，以实现条件性基因敲除、单碱基或序列替换、细胞或基因标记等多种精确和( 或) 复杂的基因组靶向修饰。通过长同源臂介导的同源重组(HR) 和微同源序列介导的同源修复两种途径可实现精确的靶向敲入，非同源末端连接(NHEJ) 途径则可介导非精确的靶向敲入，或称靶向插入。一般而言，基于同源序列的精确敲入的效率低于NHEJ 诱导的靶向插入。受益于以TALEN、CRISPR/Cas 系统为代表的人工核酸内切酶介导的基因组编辑技术的飞速发展，基因组靶向敲入得以在多个物种中越来越广泛地应用，并极大地推动了基因功能与疾病模型研究。不过，在个体水平上，相比于简单的indel 突变，基因组靶向敲入仍较难实现，效率仍然偏低。现介绍基因组靶向敲入的基本原理，并以斑马鱼为例，介绍基因组靶向敲入在模式动物中的应用与相关技术的发展历程，并着重强调实验设计与操作的要点，同时对基因组靶向敲入技术的发展进行了展望。
Abstract: Abstract: Targeted knock-in (KI) refers to the experimental methods to introduce exogenous DNA sequence into a specific site to achieve genome modification such as conditional knock-out (KO)， nucleotide or fragment substitution， cell or gene labeling， etc. Precise KI could be achieved via traditional long-homology arm-directed homologous recombination (HR) or micro-homology based DNA repair， while imprecise KI could be generated through non-homologous end joining (NHEJ)-mediated targeted insertion， which is generally higher in efficiency than the homology-based precise KI. In recent years， following the revolution of genome editing technology brought by the fast development of engineered endonucleases including TALEN， CRISPR/Cas system， KI strategy becomes more and more popular in many model organisms， which greatly facilitated the study of gene function and disease modeling. However， the efficiency of knock-in is still much lower than knock-out at individual level， and the technique practice is still challenging for most researchers. Here， using zebrafish as a major model， we briefly summarized the basic principle and progress of knock-in approaches， as well as experimental design and technical considerations. We also discussed the future directions of this important yet challenging technology.