靶向食蟹猴NTCP基因的CRISPR/Cas9系统gRNA筛选

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。

gRNA Screening of CRISPR/Cas9 System Targeting Macaca fascicularis NTCP Gene

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 165 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 transferred,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 activity to make a frame-shift mutation in NTCP gene.This study laid a theoretical foundation for further editing NTCP gene and its biological function in Macaca fascicularis.