利用CRISPR/Cas9系统建立斑马鱼Spag6基因敲除模型

Establishment of Sperm-Associated Antigen 6(Spag6)Gene Knockout Zebrafish Model with CRISPR/Cas9 System

目的采用高效基因编辑系统CRISPR/Cas9对斑马鱼的精子相关抗原6(Spag6)基因进行编辑,建立Spag6基因敲除斑马鱼模型。方法针对斑马鱼Spag6基因,利用生物信息学选取靶位点,构建向导RNA(gRNA)表达载体并体外转录,将gRNA和Cas9mRNA混合物显微注射到斑马鱼单细胞期的受精卵中,24~48h提取基因组DNA,PCR扩增出目的片段,限制性酶切法初步检测基因打靶情况,最后进行测序分析确定编辑效果。结果取3个靶位点(S1、S2、S3)进行实验,选择gRNA浓度较高的S1和S3进行注射。限制性酶切及测序的结果显示S3已产生编辑效应。结论通过CRISPR/Cas9系统成功地获得Spag6基因编辑的突变体,为进一步探讨斑马鱼Spag6基因的体内功能奠定了基础。

Objective To establish a sperm-associated antigen 6(Spag6)gene knockout zebrafish model using CRISPR/Cas9 technology,an efficient gene editing system.Methods The Spag6-targeting regions were chosen by bioinformatics analysis and the gRNA expression vectors established and transcribed in vitro.Approximately 1 nL of Cas9 RNA and gRNA mixture was co-injected into each yolk of one-cell stage embryo.Genomic DNA was extracted after 24-48 h,and the target fragment was amplified by PCR.Editing effect was evaluated by digestion of the PCR products and DNA sequencing.Results Three gRNAs were synthesized(named S1,S2,S3),and the two with higher concentrations(S1 and S3)were injected.Restriction enzyme digestion and DNA sequencing revealed that the Spag6 gene was successfully edited by S3.Conclusion Spag6 gene of zebrafish was successfully edited by CRISPR/Cas9 system,which provides a basis for further study of Spag6 gene in vivo.