利用CRISPR/Cas9系统构建TiKi1和TiKi2双基因敲除兔

Buiding a TiKi2 and TiKi1 knockout rabbit model using the CRISPR/Cas9 system

Tiki基因为新发现的基因,在蛙上的功能研究证实Tiki在头部诱导的过程中起着决定性的作用。由于TiKi基因包含TiKi2和TiKi1两种类型,因此建立一种同时敲除TiKi2和TiKi1两种基因的兔模型,能更好地研究TiKi基因是否影响兔头部的早期发育。本研究通过CRISPR/Cas9系统结合原核期受精卵RNA胞质内显微注射来建立家兔双基因打靶技术体系。考虑到TiKi1和TiKi2的同源性比较高,可能相互之间存在补偿效应,于是设计了一个gRNA能同时靶向TiKi1和TiKi2。将200 ng/μL Cas9 mRNA和20 ng/μL gRNA注射到51个处于原核期的家兔受精卵的胞质内,移植到6只受体兔体内,共计生出12只仔兔,经测序鉴定其中有1只仔兔为TiKi1和TiKi2双等位基因敲除模型。结果表明CRISPR/Cas9系统对于家兔双基因修饰研究是一种高效和简便的工具,成功建立的TiKi1和TiKi2双基因敲除兔模型在评估新药疗效、基因治疗等的研究领域中将具有很大的应用潜力。

Tiki gene is a newly discovered gene.Functional studies on frogs have confirmed that Tiki plays a decisive role in the process of head induction.Since the TiKi gene contains two types of TiKi2 and TiKi1,It is better to establish a rabbit model that simultaneously knocks out the two genes TiKi2 and TiKi1 in order to better study whether the TiKi gene affects the early development of the rabbit head.This study was to establish a target system of rabbit double gene by CRISPR/Cas9 system combinedwith intracellular microinjection of prokaryotic fertilized egg RNA.Considering that the homology of TiKi1 and TiKi2 is relatively high,there may be compensatory effects between each other,so we designed a gRNA to simultaneously target TiKi1 and TiKi2.In this study,200 ng/μL Cas9 mRNA and 20 ng/μL gRNA were injected into the cytoplasm of 51 fertilized eggs of rabbits in the prokaryotic stageand transplanted into 6 recipient rabbits.A total of 12 rabbits were born,After sequencing,one of the rabbits was identified as TiKi1 and TiKi2 double allele knockout models.The results indicate that the CRISPR/Cas9 system is an efficient and simple tool for rabbit double gene modification studies.The TiKi1 and TiKi2 double knockout rabbit models successfully established in this study will have great application potential in the research field of evaluating new drug efficacy and gene therapy.