人抑癌基因p53与PTEN的CRISPR-Cas9导向RNA靶点筛选与效率验证

doi:10.13481/j.1671-587x.20180206

摘要/Abstract

摘要： 目的：探讨应用新兴基因编辑技术规律性成簇的间隔短回文重复序列-核酸酶系统（CRISPR-Cas9）对人体内2个抑癌基因p53与PTEN进行编辑的可行性，并通过体外检测研究其编辑效果，为原代细胞向肿瘤细胞转化及建立人源化小鼠肿瘤模型提供实验研究工具。方法：序列分析确定p53及PTEN基因各亚型mRNA剪辑的共同外显子区域，针对该区域进行软件评分，设计选择针对p53及PTEN基因的CRISPR-Cas9导向RNA （sgRNA）靶点各2个，即p53-1、p53-2、PTEN-1和PTEN-2。构建共表达sgRNA与Cas9-P2A-GFP的CRISPR-Cas9质粒。选择处于对数生长期的293T细胞，将其分为对照组（未转染质粒的野生型293T细胞）和实验组（利用Lipofectamine2000转染sgRNA-Cas9-P2A-GFP共表达质粒的293T细胞），流式细胞术分选实验组绿色荧光蛋白（GFP）阳性细胞（成功转染质粒的细胞），2周扩增培养后提取基因组DNA，PCR扩增基因组DNA包含突变位点的区段，将扩增产物凝胶电泳后胶回收，连接至pEASY-Blunt Zero克隆载体中，转化，挑取单克隆菌斑培养，提取质粒DNA测序，对比对照组与实验组测序结果后确定RNA介导的特异基因位点突变效率。结果：转染sgRNA-Cas9-P2A-GFP质粒的实验组流式细胞术分选后GFP阳性率高达82%，与分选前（4%）比较明显升高（P<0.05）。对分选培养后的细胞提取基因组PCR，其中包含p53突变位点的扩增片段长度均为612 bp，而包含PTEN-1突变位点的扩增片段长度为667 bp，包含PTEN-2突变位点的扩增片段长度为947 bp。对比实验组基因测序结果与未经sgRNA-Cas9-P2A-GFP质粒转染的对照组野生型细胞测序结果，p53-1、p53-2和PTEN-2诱导的突变效率分别为54.5%、45.5%和33.3%。结论：针对人p53及PTEN基因设计的sgRNA p53-1、p53-2和PTEN-2能够在基因组水平较高效率地成功介导Cas9进行位点特异性的基因编辑。

关键词: 抑癌基因p53, 抑癌基因PTEN, 规律性成簇的间隔短回文重复序列, 导向RNA, 人类

Abstract: Objective:To explore the feassibility to edit human p53 and PTEN, two tumor suppressor genes, by CRISPR-Cas9 technology and to evaluate the editing efficiency in vitro,and to provide the experimental study tools for transforming primary healthy cells into malignant tumor cells and establishment of humanized mouse models with human oncogenesis in vivo. Methods: The single-guide RNA (sgRNA) sequences were designed to target the common exon regions of p53 and PTEN mRNA isoforms based on software analysis, that could predict their gene editing efficiency. The sgRNAs with high scores were selected and cloned into Cas9-P2A-GFP plasmid to construct sgRNA-Cas9-P2A-GFP vector that co-expressed sgRNA, Cas9 and GFP. The 293T cells in logarithmic growth phase were transfected with the sgRNA-Cas9-P2A-GFP vector (experimental group) or PBS (control group) by Lipofectamine 2000. After two-week expansion, the GFP-positive 293T cells were purified by flow cytometric sorter, whose genomic DNA was extracted for further analysis. The DNA fragments containing the sgRNA targeting site were amplified from the extracted genomic DNA by PCR and purified by gel extraction. Then they were linked into the pEASY-Blunt Zero cloning vector and transformed into competent E. coli cells. The single colonies formed by pEASY-Blunt Zero vector transformed cells were used to extract the plasmid for DNA sequencing. And the sequencing results of control group and experimental group were compared to judge the gene editing efficiency. Results: Over 82% of the sgRNA-Cas9-P2A-GFP transfected cells were found to express GFP gene after flow sorting in experimental group, which was significantly higher than that of the pre-sorted cells (P<0.05). Genomic DNA was extracted from the sorted cells after expansion and used as PCR template. The length of the amplified fragments containing the p53 mutation site was 612 bp, while the lengths of the amplified fragments containing the PTEN-1/PTEN-2 mutation site were 667 and 947 bp. The results of sequencing showed that the efficiency of editing induced by p53-1, p53-2 and PTEN-2 sgRNA were 54.5%, 45.5% and 33.3%, respectively. Conclusion: The sgRNAs p53-1, p53-2 and PTEN-2 designed for p53 and PTEN can successfully guide Cas9-mediated site-specific genome editing with high efficiency at the genome level.

Key words: single-guide RNA, PTEN, CRISPR, p53, human

中图分类号:

许文舒, 胡正, 杨永广, 王晓丹. 人抑癌基因p53与PTEN的CRISPR-Cas9导向RNA靶点筛选与效率验证[J]. 吉林大学学报(医学版), 2018, 44(02): 235-242.

XU Wenshu, HU Zheng, YANG Yongguang, WANG Xiaodan. Screening and efficiency verification of CRISPR-Cas9 single-guide RNA against human tumor suppressor gene p53 and PTEN[J]. Journal of Jilin University Medicine Edition, 2018, 44(02): 235-242.

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