基于新的CRISPR/Cas9技术实现斑马鱼LHX9基因的快速编辑及对F0突变体性腺发育的初筛

Using A newly Rapid CRISPR/Cas9 System to Perform Genome Editing of Zebrafish LHX9 Gene and Preliminary Screening Gonad Development in F0 Mutant

目的:CRISPR/Cas9系统在斑马鱼的反向遗传学中的到了广泛应用,但突变基因的表型观察往往需要在突变鱼系的F1中进行,费时较长。LHX9作为LIM家族的一种转录因子,在胚胎早期的泌尿生殖嵴中有广泛分布;且LHX9基因敲除的小鼠存在性腺发育不良。本研究拟通过一种新的CRISPR/Cas9基因编辑技术,采用四条sgRNA对LHX9基因进行VASA转基因斑马鱼的基因敲除,以观察该基因缺陷对斑马鱼性腺发育的影响。方法:利用新的CRISPR/Cas9技术,设计四条针对斑马鱼LHX9基因3号外显子的20bp的sgRNA,通过非克隆体外转录得到靶位点的四条sgRNA。将以上靶位点的四条sgRNA与Cas9核酸酶蛋白同时注射入单细胞期的斑马鱼胚胎内,利用PCR鉴定突变型类型和突变比例。通过对LHX9基因突变体的VASA转基因斑马鱼进行荧光观察,发现LHX9基因缺陷的斑马鱼性腺发育的情况。结果:靶向Exon 3的四条sgRNA可成功编辑斑马鱼LHX9基因,敲除效率高达82%,Sanger测序发现产生10种不同的移码突变类型。通过该方法对VASA转基因斑马鱼的LHX9基因进行编辑,发现LHX9基因突变导致dph6的的斑马鱼原始生殖细胞增殖和迁移受到影响。结论:基于4条sgRNA注射的CRISPR/Cas9技术,可以快速地产生具有突变表型的G0斑马鱼,具有应用潜力。LHX9基因敲除导致原始生殖细胞的发育和迁移受到影响,提示该基因参与了斑马鱼早期性腺的发育。

Objective:CRISPR/Cas9 system has been widely used in reverse genetics of zebrafish,but it often takes longer time to observe and screen phenotype of mutant genes in F1 of mutant fish lines.LHX9,the LIM homeobox gene,is widely expressed in urogenital ridges in early embryonic stage.In LHX9 knockout mice,somatic cells of the genital ridge fail to proliferate,leading to disturbence of gonads formation.In this study,four sgRNAs were designed to target the LHX9 gene in VASA transgenic zebrafish by a new CRISPR/Cas9 gene editing technique to rapidly observe the effect of LHX9 knockout in the gonad development of zebrafish.Methods:Four 20 bp sgRNAs targeting exon 3 of zebrafish LHX9 gene were designed.These sgRNAs were obtained by non-cloning in vitro transcription.The mixture of four sgRNAs were injected into VASA transgenic zebrafish embryos at single cell stage together with Cas9 nuclease.The mutation types and mutation proportions were identified by PCR and sequencing.The development of gonads in zebrafish with LHX9 gene defect was observed by fluorescence microscopy in VASA transgenic zebrafish.Results:Four sgRNAs targeting Exon 3 could successfully edit the LHX9 gene of zebrafish.The knockout efficiency was as high as 82%,and Sanger sequencing revealed 10 different types of shifting mutations.In a VASA transgenic zebrafish,it was found that the proliferation and migration of primordial germ cells were affected if LHX9 gene was disturbed.Conclusions:CRISPR/Cas9 technology based on four sgRNA injections can rapidly produce F0 zebrafish with mutant phenotype and has potential application.The knockout of LHX9 gene affect the proliferation and migration of primordial germ cells,suggesting its role in the early development of gonads in zebrafish.