利用CRISPR/Cpf1系统构建人ABCG1和ABCG4基因敲除的293T细胞株
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摘要
【目的】基于CRISPR/Cpf1(AsCpf1/LbCpf1)技术构建人ABCG1和ABCG4基因敲除的人胚肾细胞(HEK293T)稳定细胞株,旨在研究ABCG1和ABCG4基因的生物学功能。【方法】针对ABCG1和ABCG4基因作用的功能域,设计2对靶向ABCG1和ABCG4基因前7个外显子的sgRNA序列,分别克隆到AsCpf1和LbCpf1的载体上。将测序验证正确的重组质粒转染到HEK293T细胞中,T7E1酶切和测序验证敲除效率,对敲除成功的293T细胞利用有限稀释法筛选获得基因双突变的敲除细胞株,对其基因组进行PCR扩增并双向测序验证编辑结果,选择有正确编辑结果的PCR产物进行TA克隆,进而测序验证单克隆细胞株编辑效率。【结果】结果获得敲除ABCG1和ABCG4基因的稳定细胞株各1株(AsCpf1-ABCG1-sgRNA 1-1和LbCpf1-ABCG4-sgRNA 2-1)。【结论】通过利用新型基因编辑技术CRISPR/Cpf1系统,获得了永久性敲除目的细胞靶基因的细胞株,可为进一步探索和证实ABCG1和ABCG4在细胞中胆固醇代谢和调节作用提供帮助。
【Objective】To study the biological function of ABCG1 and ABCG4 genes, we constructed human stable HEK293 T cell line of ABCG1 and ABCG4 genes knockout through CRISPR/Cpf1 system. 【Method】Two small guide RNA(sgRNA) targeting the first to seven exons of ABCG1 and ABCG4 CDS regions to the functional domains of ABCG1 and ABCG4 genes were designed and cloned into AsCpf1 and LbCpf1 vectors, respectively. The sequencing-verified recombinant plasmids were transfected into HEK293 T cells, and the knockout cell lines with double gene mutations were screened by limiting dilution method. 【Result】The results showed that both ABCG1 and ABCG4 biallelic alleles had insertions or deletions of bases at the target site, and ABCG1(1 strain) and ABCG4(1 strains) mutant cell lines were successfully obtained. 【Conclusion】A cell line that permanently knocks out the target gene of the target cell has been obtained, which can help to further explore and confirm the cholesterol metabolism and regulation of ABCG1 and ABCG4 in the cell.
【Objective】To study the biological function of ABCG1 and ABCG4 genes, we constructed human stable HEK293 T cell line of ABCG1 and ABCG4 genes knockout through CRISPR/Cpf1 system. 【Method】Two small guide RNA(sgRNA) targeting the first to seven exons of ABCG1 and ABCG4 CDS regions to the functional domains of ABCG1 and ABCG4 genes were designed and cloned into AsCpf1 and LbCpf1 vectors, respectively. The sequencing-verified recombinant plasmids were transfected into HEK293 T cells, and the knockout cell lines with double gene mutations were screened by limiting dilution method. 【Result】The results showed that both ABCG1 and ABCG4 biallelic alleles had insertions or deletions of bases at the target site, and ABCG1(1 strain) and ABCG4(1 strains) mutant cell lines were successfully obtained. 【Conclusion】A cell line that permanently knocks out the target gene of the target cell has been obtained, which can help to further explore and confirm the cholesterol metabolism and regulation of ABCG1 and ABCG4 in the cell.
引文
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[10]刘玉彪,许馨,曹山虎,等.基因编辑技术最新研究进展[J].生物技术通报,2017,33(6):39-44.
[2]杨婷,王苏梦,汪俊军.ABCG1在胆固醇转运中作用研究新进展[J].医学研究生学报,2015(1):89-93.
[3]李瞿,张小倩,刘旭,等.ATP结合盒转运体G1与动脉粥样硬化的研究进展[J].中国医科大学学报,2017,46(4):289-293.
[4]Hegyi Z,Homolya L.Functional Cooperativity between ABCG4 and ABCG1 Isoforms[J].Plos One,2016,11(5):e0156516.
[5]Hsu P D,Lander E S,Zhang F.Development and applications of CRISPR-Cas9 for genome engineering[J].Cell,2014,157(6):1262–1278.
[6]Jinek M,Chylinski K,Fonfara I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337(6096):816-821.
[7]杨帆,李寅.新一代基因组编辑系统CRISPR/Cpf1[J].生物工程学报,2017,33(3):361-371.
[8]Ran F A.Adaptation of CRISPR nucleases for eukaryotic applications[J].Analyt Biochem,2016,doi:10.1016/j.ab.2016.10.018.(in Press)
[9]Hung SSC,McCaughey T,Swann O,et al.Genome engineering in ophthalmology:application of CRISPR/Cas to the treatment of eye disease[J].Prog Retin Eye Res,2016,53:1-20.
[10]刘玉彪,许馨,曹山虎,等.基因编辑技术最新研究进展[J].生物技术通报,2017,33(6):39-44.