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

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

Abstract

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

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.

References

[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.