CRISPR/Cas9系统介导大鼠Inhba基因的编辑

Inhba gene editing mediated by CRISPR/Cas9 system in rat

利用chopchop网站对大鼠Inhba基因的第二外显子设计sgRNA位点,通过合成sgRNA寡核苷酸、酶切连接构建到px330载体,再转染px330-Inhba-sgRNA载体到大鼠L6细胞,通过T7E1酶切、T载体克隆测序来验证sgRNA编辑Inhba基因的效率。结果表明:转染pX330-Inbha-sgRNA载体后,Inhba基因的编辑区域的PCR产物能被T7E1酶切割预期条带;T载体克隆测序显示,随机选取的20个单克隆中有5个克隆在预期切割位点附近出现不同长度的碱基缺失,估测编辑效率为25%。可见,本研究设计的sgRNA能够有效利用CRISPR/Cas9系统对Inhba基因进行编辑。

The sgRNA site was designed by the chopchop website on the second exon of the rat Inhba gene and was constructed into the px330 vector by synthesizing sgRNA oligonucleotides and enzyme digestion and ligation.The px330-Inhba-sgRNA vector was transfected into rat L6 cells,and the efficiency of sgRNA on Inhba gene editing was verified by T7E1 digestion and T vector cloning and sequencing.The results showed that after transfection of the pX330-Inbha-sgRNA vector,the PCR product in the editing region of the Inhba gene could be cleaved by the T7E1 enzyme to produce the expected fragment.T vector cloning and sequencing showed that five in the selected randomly twenty clones showed different lengths of base deletion at the targeted cutting sites,and the estimated editing efficiency was 25%.In summary,it showed that the sgRNA designed in this study could effectively edit the Inhba gene using the CRISPR/Cas9 system in rat.