构建基于CRISPR/Cas9技术敲除ALOX5的质粒

doi:10.13560/j.cnki.biotech.bull.1985.2019-0270

生物技术通报 ›› 2020, Vol. 36 ›› Issue (3): 110-114.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0270

• 基因编辑专题（专题主编谷峰研究员） • 上一篇下一篇

构建基于CRISPR/Cas9技术敲除ALOX5的质粒

梅芬^{1, 2}, 李瑞玮^{1, 3}, 张娟娟¹, 左荣霞¹, 邹云莲¹, 沈涛¹, 撒亚莲¹

1. 昆明理工大学附属医院云南省第一人民医院临床基础医学研究所云南省出生缺陷与遗传病研究重点实验室云南省临床病毒学重点实验室,昆明 650032;
2. 华中科技大学同济医学院生殖医学中心（武汉同济生殖医学专科医院）,武汉 430030;
3. 云南省第三人民医院生殖遗传科,昆明 650011

收稿日期:2019-04-08 出版日期:2020-03-26 发布日期:2020-03-17
作者简介:梅芬,女,硕士研究生,研究方向：疾病相关基因;E-mail：752395090@qq.com;李瑞玮为共同第一作者
基金资助:
国家自然科学基金项目（31460298）,云南省卫生厅内设机构资助项目（2016NS225）,云南省科技厅-昆医联合专项（2015FB102）

Construction of Plasmids for Knocking out ALOX5 Gene Using CRISPR/Cas9 Technology

MEI Fen^{1, 2}, LI Rui-wei^{1, 3}, ZHANG Juan-juan¹, ZUO Rong-xia¹, ZOU Yun-lian¹, SHEN Tao¹, SA Ya-lian¹

1. Institute of Clinical and Basic Medical Sciences,The First People’s Hospital of Yunnan Province（Yunnan Provincial Key Laboratory of Clinical Virology,Key Laboratory for Birth Defects and Genetic Diseases）,Kunming 650032;
2. Center of Reproductive Medicine,Tongji Medical College（Reproduce Medicine Hospital of Tongji Medical College）,Huazhong Science and Technological University,Wuhan 430030;
3. Department of Reproductive Genetics,The Third People’s Hospital of Yunnan Province,Kunming 650011;

Received:2019-04-08 Published:2020-03-26 Online:2020-03-17

摘要/Abstract

摘要：

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因（Arachidonate 5-lipoxygenase gene,ALOX5）的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5 基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5 基因的重组质粒pX458-sgRNAs-ALOX5。

关键词: ALOX5, CRISPR/Cas9, 基因敲除, 构建, 质粒

Abstract:

This work aims to construct the plasmids targeting for ALOX5 gene knockout by CRISPR/Cas9 technology. Three pairs of oligonucleotides sgRNAs targeting exon 6 of ALOX5 gene were designed,chemically synthesized,and inserted into linearized plasmids pX458,respectively. The recombinant plasmids pX458-sgRNAs-ALOX5 was then transformed into competent Escherichia coli DH5α. Whether or not recombinant plasmids were constructed successfully was evaluated by Sanger sequencing. The constructed recombinant plasmids were transfected into 293T cells,and the transfection efficiency was observed under fluorescence microscope. Then their genomic DNA derived from the successfully-transfected cells were extracted by the reagent kit. The DNA fragment with target gene knockout was amplified by PCR,the nucleotides were obtained by sequencing,and the knockout of ALOX5 gene in the transfected cells was analyzed by DNAStar software. The sequencing results revealed that 2 pairs of double stranded sgRNA oligodeoxynucleotides were successfully inserted into the plasmids with correct sequences,and the construction of recombinant plasmid pX458-sgRNAs-ALOX5 targeting ALOX5 gene was successful. The transfection efficiency of pX458-sgRNAs-ALOX5 in 293T was about 50%;however,cleaving effect was not detected by Sanger sequencing. In summary,the CRISPR/Cas9-based plasmids pX458-sgRNAs targeting the exon 6 of ALOX5 gene is successfully constructed.

Key words: ALOX5, CRISPR/Cas9, gene knock out, construction, plasmids

梅芬, 李瑞玮, 张娟娟, 左荣霞, 邹云莲, 沈涛, 撒亚莲. 构建基于CRISPR/Cas9技术敲除ALOX5的质粒[J]. 生物技术通报, 2020, 36(3): 110-114.

MEI Fen, LI Rui-wei, ZHANG Juan-juan, ZUO Rong-xia, ZOU Yun-lian, SHEN Tao, SA Ya-lian. Construction of Plasmids for Knocking out ALOX5 Gene Using CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2020, 36(3): 110-114.

参考文献

[1] 黄凯, 蒋世云, 陈柳军. 5-脂氧合酶及其抑制剂[J]. 中国生物化学与分子生物学报, 2014, 30(12):1190-1196.
[2] Chen Y, Hu Y, Zhang H, et al.Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia[J]. Nat Genet, 2009, 41(7):783-792.
[3] Chen Y, Peng C, Sullivan C, et al.Critical molecular pathways in cancer stem cells of chronic myeloid leukemia[J]. Leukemia, 2010, 24(9):1545-1554.
[4] Roos J, Oancea C, et al.5-Lipoxygenase is a candidate target for therapeutic management of stem cell-likecells in acute myeloid leukemia[J]. Cancer Res, 2014, 74(18):5244-5255.
[5] Lone BA, Karna SKL, et al.CRISPR/Cas9 system:A bacterial tailor for genomic engineering[J]. Genet Res Int, 2018:3797214.
[6] Naert T, Vleminckx K.Genotyping of CRISPR/Cas9 genome edited Xenopus tropicalis[J]. Methods Mol Biol, 2018, 1865:67-82.
[7] 贾启鹏, 申培磊, 张欢, 等. CRISPR/Cas9系统介导敲除CSN2基因奶山羊胎儿成纤维突变细胞的制备[J]. 生物技术通报, 2017, 33(9):131-138.
[8] 马晓丽, 景巧丽, 裴雁曦. 基于CRISPR/Cas9技术的大白菜内源硫化氢生成酶LCD基因突变体构建[J]. 中国细胞生物学学报, 2018, 40(11):1799-1805.
[9] 姚恒, 杨大海, 白戈, 等. 利用CRISPR/Cas9技术定点敲除烟草多酚氧化酶基因NtPPO1[J]. 生物技术通报, 2018, 34(11):97-102.
[10] 唐石树, 胡建安. 5-脂氧合酶在人体组织中的表达及其与疾病的关系[J]. 中南大学学报:医学版, 2015, 40(4):438-445.
[11] Mashima R, Okuyama T.The role of lipoxygenases in pathophysiology;new insights and future perspectives[J]. Redox Biol, 2015, 6:297-310.
[12] Che XH, Chen CL, Ye XL, et al.Dual inhibition of COX-2/5-LOX blocks colon cancer proliferation, migration and invasion in vitro[J]. Oncol Rep, 2016, 35(3):1680-1688.
[13] Sarveswaran S, Varma N, Morisetty S, et al.Inhibition of 5-lipoxygenase downregulates stemness and kills prostate cancer stem cells by triggering apoptosis via activation of c-Jun N-terminal kinase[J]. Oncotarget, 2016, 17.
[14] Bishayee K, Khuda-Bukhsh AR.5-lipoxygenase antagonist ther-apy:a new approach towards targeted cancer chemotherapy[J]. Acta Biochim Biophys Sin, 2013, 45(9):709-719.
[15] Anderson KM, Seed T, et al.Selective inhibitors of 5-lipoxygenase reduce CML blast cell proliferation and induce limited differentia-tion and apoptosis[J]. Leuk Res, 1995, 19(11):789-801.
[16] Rådmark OP.The molecular biology and regulation of 5-lipoxyge-nase[J]. Am J Respir Crit Care Med, 2000, 161(2):S11-S15.
[17] Lo MC, Peterson LF, Yan M, et al.Combined gene expression and DNA occupancy profiling identifies potential therapeutic targets of t(8;21)AML[J]. Blood, 2012, 120(7):1473-1484.
[18] Woessner DW, Lim CS.Disrupting BCR-ABL in combination with secondary leukemia-specific pathways in CML cells leads to enhanced apoptosis and decreased proliferation[J]. Mol Pharm, 2013, 10(1):270-277.
[19] Hu Y, Chen Y, Douglas L, et al.beta-Catenin is essential for survival of leukemic stem cells insensitive to kinase inhibition in mice with BCR-ABL-induced chronic myeloid leukemia[J]. Leukemia, 2009, 23(1):109-116.
[20] Wang Y, Krivtsov AV, Sinha AU, et al.The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML[J]. Science, 2010, 327(5973):1650-1653.
[21] 雒钰杰, 徐敏, 高雯琬, 等. 敲低花生四烯酸5脂加氧酶(Alox5)基因促进K562/ADM细胞凋亡[J]. 细胞与分子免疫学杂志, 2017, 33(10):1398-1403.
[22] 王大勇, 马宁, 惠洋, 等. CRISPR/Cas9基因组编辑技术在癌症研究中的应用[J]. 遗传, 2016, 38(1):1-8.
[23] Ran FA, Hsu PD, et al.Genome engineering using the CRISPR-Cas9 system[J]. Nat Protoc, 2013, 8(11):2281-2308.

构建基于CRISPR/Cas9技术敲除ALOX5的质粒

Construction of Plasmids for Knocking out ALOX5 Gene Using CRISPR/Cas9 Technology

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