基于CRISPR/Cas9系统构建青鳉突变体

Establishment of Japanese medaka(Oryzias latipes)mutants based on CRISPR/Cas9 system

青鳉是研究基因功能、器官发生和发育机制的模式生物之一,基因编辑技术已在青鳉中得到广泛应用,但利用CRISPR/Cas9技术构建青鳉突变体的详细过程还未见报道。为了完整详细地阐明青鳉突变体的构建过程,实验以青鳉Olpax6.1基因的敲除为例,首先利用ChopChop网站设计获得Olpax6.1基因gRNA的靶点;其次通过PCR扩增和质粒酶切分别获得gRNA和Cas9 mRNA体外转录的模板,并通过体外转录合成gRNA和Cas9 mRNA;再将Cas9 mRNA和gRNA混合进行青鳉胚胎显微注射获得突变F_(0);最后利用PCR、T7EndonucleaseⅠ酶切和Sanger测序筛选F_(0)与野生型杂交的子代获得相同突变类型的F_(1),进而将相同突变的F_(1)进行杂交并筛选其子代,获得青鳉Olpax6.1敲除的纯合突变体,纯合突变体表现出眼部发育畸形的经典表型。本研究为青鳉突变体的构建提供详细完整的实验方案,同时也为其他鱼类突变体的构建提供技术参考。

Medaka(Oryzias latipes)is one of the model organisms for studying gene function,organogenesis,and development mechanism.Although editing technology has been widely used in medaka,there was no detailed report on the use of CRISPR/Cas9 to establish O.latipes mutant.To thoroughly describe the establishment process of O.latipes mutant,this study takes the knockout of Olpax6.1 as an example.Firstly,the gRNA target of Olpax 6.1 gene was designed using the ChopChop website;secondly,the template for in vitro transcription of gRNA and Cas9 mRNA were obtained by PCR and plasmid digestion respectively,and gRNA and Cas9 mRNA were synthesized by in vitro transcription;thirdly,the mixture of Cas9 mRNA and gRNA was microinjected into medaka embryos to obtain the mutated F_(0);finally,we used PCR,T7 Endonuclease I digestion and Sanger sequencing to screen the offspring of the cross between F_(0) and wild type to obtain F_(1) with the same mutation type,and then crossed the same mutated F_(1) and screened their offspring to obtain Olpax6.1 knockout homozygous mutant,which displays the classic phenotype of ocular developmental malformations.Taken together,this study provides technical guidance for the generation of mutants in O.latipes and other fish.