应用Cas9 RNPs进行绵羊FecB基因型检测的可行性研究

A feasibility study of using Cas9 ribonucleoproteins for genotyping of FecB in sheep

为了研究Cas9 RNPs用于绵羊FecB基因分型的可行性,试验以FecB基因BB、B+、++型的绵羊个体为试验动物提取基因组DNA。基于Cas9 RNPs的体外DNA定点内切酶活性,用FecB基因靶位点的特异性引物扩增产物作为底物,利用大肠杆菌共表达纯化获得Cas9 RNPs,对不同FecB基因型目标样本进行酶切检测。结果表明:SDS-PAGE电泳结果显示,在130~180 kD之间有一条与预期大小相符的特异蛋白条带;3种基因型的靶DNA均被切割为185 bp和368 bp两个片段;绵羊FecB++型成纤维细胞Cas9 RNPs电转染的突变检测结果显示,纯化的两个Cas9 RNPs都具有基因组编辑活性。说明本研究获得的Cas9 RNPs在体外和细胞内都具有良好的酶活性,但未能区分绵羊FecB基因型。

In order to study the feasibility of using Cas9 RNPs for sheep,FecB genotyping genomic DNA was extracted from the individual tissue samples of sheep with FecB genotypes identified as wild type(++),heterozygous type(B+)and homozygous type(BB),respectively.Based on the in vitro targeted DNA endonuclease activity of Cas9 RNPs,the specific primers of target sites in FecB genes were used to amplify the targeted DNA fragments which would be used as substrates.Cas9 RNPs were directly prepared and purified from Escherichia coli of co-expressing Cas9 and target specific single guided RNAs.The genomic DNA from sheep with genotypes of++,B+and BB,respectively,were digested with Cas9 RNPs.The SDS-PAGE results showed that a specific protein band in the range of 130~180 kD was found consistent with the expected size.DNA fragments of both 185 bp and 368 bp were generated in all digested samples.The mutation assay of electrotransfection with Cas9 RNPs of fibroblasts with genotypes of FecB++showed that both of purified Cas9 RNPs had genome editing activities.It could be concluded that sheep FecB may not be genotyped by Cas9 RNPs obtained in this study,even showing good enzyme activity in vivo or in vitro.