靶向食蟹猴NTCP基因的CRISPR/Cas9系统gRNA筛选

摘要

This study was aimed to screen gRNA of efficient knockout activity targeting Macaca fascicularis NTCP gene by the CRISPR/Cas9 enzyme digestion method and PCR amplification methods in vitro .Comparing NTCP gene sequences between Macaca fascicularis and human,the sequences of NTCP gene coding amino acid 84 to 87 and 157 to 1 65 were chosen as gene knockout targets.3 to 4 candidate gRNA sequences were designed in two target sequence regions through gRNA software.By screening cleavage activity targeting NTCP gene in vitro ,gRNA1.2 and gRNA2.1 were selected and inserted into pLV hUbC-Cas9-T2A-GFP plasmid,respectively.The genome DNA was extracted from primary hepatocytes after gRNA1.2 and gRNA2.1 being trans-ferred,respectively.Then NTCP sequences were amplified by PCR and sequenced by being cloned into T vector.The results indicated that compared to gRNA1.2,gRNA2.1 had much higher activ-ity to make a frame-shift mutation in NTCP gene.This study laid a theoretical foundation for fur-ther editing NTCP gene and its biological function in Macaca fascicularis .%本研究旨在通过 CRISPR/Cas9体外酶切法及细胞水平上的 PCR 扩增测序筛选出靶向食蟹猴 NTCP 基因具有高敲除活性的 gRNA。首先通过比对食蟹猴与人类 NTCP 氨基酸序列,选择差异位点,即第84—87位和第157—165位氨基酸作为基因靶点序列区；利用 gRNA 软件设计针对上述基因靶点序列的 gRNA,每个靶点设计3~4条候选 gRNA 序列；然后利用 gRNA 体外检测试剂盒,筛选出靶向 NTCP 基因的体外敲除活性较高的两条gRNA 序列：gRNA1.2和 gRNA2.1。将 gRNA1.2和 gRNA2.1分别插入 pLV hUbC-Cas9-T2A-GFP 载体中,转染食蟹猴原代肝细胞。提取转染后细胞基因组 DNA,通过 PCR 扩增 NTCP 基因并将其克隆到 T 载体中进行测序分析。结果表明,gRNA1.2和 gRNA2.1均可使 NTCP 基因产生移码突变,但 gRNA2.1比 gRNA1.2具有更高的敲除活性。本研究为下一步编辑食蟹猴 NTCP 基因及研究其在乙型肝炎病毒(HBV)感染中的功能奠定了基础。