应用CRISPR/Cas9系统构建Rev-erbβ基因敲除的HEK293细胞系
|
摘要
目的利用成簇的、规律间隔的短回文重复序列/Cas9核酸酶(CRISPR/Cas9)基因组编辑技术,构建Rev-erbβ基因敲除的HEK293细胞系。方法通过单向导RNA(sgRNA)介导Cas9蛋白对目的基因靶位点DNA进行特异性的切割,然后经DNA同源重组单向导RNA或非同源末端连接方式进行修复,以实现对Rev-erbβ基因进行敲入、敲除修饰操作的目的。首先,针对Rev-erbβ基因设计4个sgRNA,经筛选选择活性较高的sgRNA1及sgRNA2用于构建p CMV-h Cas9-U6-Rev-erbβsgRNA1&sgRNA2串联载体。然后将p CMV-h Cas9-U6-Rev-erbβsgRNA1&sgRNA2和p Ad-E1/hRev-erbβdonor质粒载体共转染至HEK293细胞,通过药物筛选、克隆化及序列测序获得整合有外源供体基因片段的一条链,另一条链为片段缺失的Rev-erbβ基因完全敲除的HEK293(Rev-erbβ-/-)细胞系。最后通过用Western blot法和实时定量PCR对敲除Rev-erbβHEK293细胞系(C3-6)进行检测。结果敲除Rev-erbβ基因的HEK293细胞系中均未检测到Rev-erbβmRNA和蛋白质的表达。结论利用CRISPR/Cas9技术,成功构建了基因定点修饰和敲除的Rev-erbβ-/-HEK293细胞系,为Rev-erbβ的功能和作用机制研究提供有效工具。
Objective To prepare Rev-erbβ knockout HEK293 cells using clustered regularly interspaced short palindromic repeats/Cas 9 nuclease(CRISPR/Cas9) gene editing technology.Methods The knock-in or knockout of Rev-erbβ gene could be realized by single-guide RNA(sgRNA)-mediated Cas9 cutting of target DNA,and followed by DNA homologous recombination or non-homologous end joining-mediated DNA repair.Firstly,four sgRNAs were designed for Rev-erbβ gene.The sgRNA1 and sgRNA2 with the higher activity were respectively used to construct p CMV-h Cas9-U6-Reverbβ sgRNA1 and p CMV-h Cas9-U6-Rev-erbβ sgRNA2.Then,p CMV-h Cas9-U6-Rev-erbβ sgRNA1,p CMV-h Cas9-U6-Reverbβ sgRNA2 and p Ad5-E1/hRev-erbβ donor plasmid vectors were co-transfected into HEK293 cells.Through drug screening,cloning and sequencing,the Rev-erbβ gene-knockout HEK293(Rev-erbβ-/-) cell lines were obtained with one chain integrated with exogenous gene fragment and the other chain for deletion mutants.Finally,the HEK293(Rev-erbβ-/-)cell lines(C3-6) was detected with real-time quantitative PCR and Western blotting.Results Expression of Rev-erbβ mRNA and protein was undetectable in HEK293 Rev-erbβ-/-cell line.Conclusion Using CRISPR/Cas9 technology,the HEK293Rev-erbβ-/-cell line has been successfully constructed,which would provide an effective tool for the study on the function of Rev-erbβ.
Objective To prepare Rev-erbβ knockout HEK293 cells using clustered regularly interspaced short palindromic repeats/Cas 9 nuclease(CRISPR/Cas9) gene editing technology.Methods The knock-in or knockout of Rev-erbβ gene could be realized by single-guide RNA(sgRNA)-mediated Cas9 cutting of target DNA,and followed by DNA homologous recombination or non-homologous end joining-mediated DNA repair.Firstly,four sgRNAs were designed for Rev-erbβ gene.The sgRNA1 and sgRNA2 with the higher activity were respectively used to construct p CMV-h Cas9-U6-Reverbβ sgRNA1 and p CMV-h Cas9-U6-Rev-erbβ sgRNA2.Then,p CMV-h Cas9-U6-Rev-erbβ sgRNA1,p CMV-h Cas9-U6-Reverbβ sgRNA2 and p Ad5-E1/hRev-erbβ donor plasmid vectors were co-transfected into HEK293 cells.Through drug screening,cloning and sequencing,the Rev-erbβ gene-knockout HEK293(Rev-erbβ-/-) cell lines were obtained with one chain integrated with exogenous gene fragment and the other chain for deletion mutants.Finally,the HEK293(Rev-erbβ-/-)cell lines(C3-6) was detected with real-time quantitative PCR and Western blotting.Results Expression of Rev-erbβ mRNA and protein was undetectable in HEK293 Rev-erbβ-/-cell line.Conclusion Using CRISPR/Cas9 technology,the HEK293Rev-erbβ-/-cell line has been successfully constructed,which would provide an effective tool for the study on the function of Rev-erbβ.
引文
[1]Cong L,Ran F A,Cox D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823.
[2]Cho S W,Kim S,Kim J M,et al.Targeted genome engineering in human cells with the Cas9RNA-guided endonuclease[J].Nat Biotechnol,2013,31(3):230-232.
[3]Jinek M,Chylinski K,Fonfara I,et al.A programmable dual RNAguide DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337(6096):816-821.
[4]张伟锋.高效的基因靶向修饰技术的建立及其应用研究[D].西安:陕西师范大学,2014.
[5]Mandal P K,Ferreira L M,Collins R,et al.Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9[J].Cell Stem Cell,2014,15(5):643-652.
[6]Gonzalez F,Zhu Z,Shi Z D,et al.An iC RISPR platform for rapid,multiplexable,andinducible genome editing in human pluripotent stem cells[J].Cell Stem Cell,2014,15(2):215-226.
[7]Mali P,Yang L,Esvelt K M,et al.RNA-guided human genome engineering via Cas9[J].Science,339(6121):823-826.
[8]Wang T,Wei J J,Sabatini D M,et al.Genetic screens in human cells using the CRISPR-Cas9 system[J].Science,2014,343(6166):80-84.
[9]Hwang W Y,Fu Y,Reyon D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system[J].Nat Biotechnol,2013,31(3):227-229.
[10]Harms D W,Quadros R M,Seruggia D,et al.Mouse genome editing using the CRISPR/Cas system[J].Curr Protoc Hum Genet,2014,83:15-17.
[11]Shen B,Zhang J,Wu H,et al.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting[J].Cell Res,2013,23(5):720-723.
[12]Wang H,Yang H,Shivalila C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J].Cell,2013,153(4):910-918.
[13]Shalem O,Sanjana N E,Hartenian E,et al.Genome-scale CRISPRCas9 knockout screening in human cells[J].Science,2014,343(6166):84-87.
[14]陈芳,夏海滨.核受体Rev-erbs的研究进展[J].生命科学研究,2013,17(6):548-553.
[15]Bugge A,Feng D,Everett L J,et al.Rev-erbαand Rev-erbβcoordinately protect the circadian clock and normal metabolic function[J].Genes Dev,2012,26(7):657-667.
[16]Lam M T,Cho H,Lesch H P,et al.Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription[J].Nature,2013,498(7455):511-515.
[17]Kumar N,Solt L A,Wang Y,et al.Regulation of adipogenesis by natural and synthetic REV-ERB ligands[J].Endocrinology,2010,151(7):3015-3025.
[18]Mei C D,Ercolani L,Parodi C,et al.Dual inhibition of REV-ERBβand autophagy as a novel pharmacological approach to induce cytotoxicity in cancer cells[J].Oncogene,2015,34(20):2597-2608.
[19]Umov F D,Rebar E J,Holmes M C,et al.Genome editing with engineered zinc finger nucleases[J].Nat Rev Genet,2010,11(9):636-646.
[20]Li T,Huang S,Zhao X,et al.Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes[J].Nucleic Acids Res,2011,39(14):6315-6325.
[21]Ji Q,Fischer A L,Brown C R,et al.Engineered zinc-finger transcription factors activate OCT4(POU5F1),SOX2,KLF4,c-MYC(MYC)and miR302/367[J].Nucleic Acids Res,2014,42(10):6158-6167.
[22]Sommer D,Peters A,Wirtz T,et al.Efficient genome engineering by targetedhomologous recombination in mouse embryos using transcription activator-like effector nucleases[J/OL].Nat Commun,2014,5:3045.doi:10.1038/ncomms4045.
[23]Cho S W,Kim S,Kim Y,et al.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases[J].Genome Res,2014,24(1):132-141.
[24]Konermann S,Brigham M D,Trevino A E,et al.Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex[J].Nature,2015,517(7536):583-588.
[25]Xiao D,Zhang W,Li Y,et al.A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene[J].J Biotechnol,2015,219:110-116.
[26]Sato S,Sakurai T,Ogasawara J,et al.A circadian clock gene,Rev-erbα,modulates the inflammatory function of macrophages through the negative regulation of Ccl2 expression[J].J Immunol,2014,192(1):407-417.
[27]Nam D,Chatterjee S,Yin H,et al.Novel function of Rev-erbαin promoting brown adipogenesis[J/OL].Sci Rep,2015,5:11239.doi:10.1038/srep11239.
[28]Wang W,Zhang Q,Zhao R,et al.Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2015,31(9):1175-1178.
[29]黄红艳,张彦斌,王小利,等.靶向敲低蛋白1抑制Hep G2肝癌细胞的增殖及迁移[J].细胞与分子免疫学杂志,2015,31(6):782-786.
[2]Cho S W,Kim S,Kim J M,et al.Targeted genome engineering in human cells with the Cas9RNA-guided endonuclease[J].Nat Biotechnol,2013,31(3):230-232.
[3]Jinek M,Chylinski K,Fonfara I,et al.A programmable dual RNAguide DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337(6096):816-821.
[4]张伟锋.高效的基因靶向修饰技术的建立及其应用研究[D].西安:陕西师范大学,2014.
[5]Mandal P K,Ferreira L M,Collins R,et al.Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9[J].Cell Stem Cell,2014,15(5):643-652.
[6]Gonzalez F,Zhu Z,Shi Z D,et al.An iC RISPR platform for rapid,multiplexable,andinducible genome editing in human pluripotent stem cells[J].Cell Stem Cell,2014,15(2):215-226.
[7]Mali P,Yang L,Esvelt K M,et al.RNA-guided human genome engineering via Cas9[J].Science,339(6121):823-826.
[8]Wang T,Wei J J,Sabatini D M,et al.Genetic screens in human cells using the CRISPR-Cas9 system[J].Science,2014,343(6166):80-84.
[9]Hwang W Y,Fu Y,Reyon D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system[J].Nat Biotechnol,2013,31(3):227-229.
[10]Harms D W,Quadros R M,Seruggia D,et al.Mouse genome editing using the CRISPR/Cas system[J].Curr Protoc Hum Genet,2014,83:15-17.
[11]Shen B,Zhang J,Wu H,et al.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting[J].Cell Res,2013,23(5):720-723.
[12]Wang H,Yang H,Shivalila C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J].Cell,2013,153(4):910-918.
[13]Shalem O,Sanjana N E,Hartenian E,et al.Genome-scale CRISPRCas9 knockout screening in human cells[J].Science,2014,343(6166):84-87.
[14]陈芳,夏海滨.核受体Rev-erbs的研究进展[J].生命科学研究,2013,17(6):548-553.
[15]Bugge A,Feng D,Everett L J,et al.Rev-erbαand Rev-erbβcoordinately protect the circadian clock and normal metabolic function[J].Genes Dev,2012,26(7):657-667.
[16]Lam M T,Cho H,Lesch H P,et al.Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription[J].Nature,2013,498(7455):511-515.
[17]Kumar N,Solt L A,Wang Y,et al.Regulation of adipogenesis by natural and synthetic REV-ERB ligands[J].Endocrinology,2010,151(7):3015-3025.
[18]Mei C D,Ercolani L,Parodi C,et al.Dual inhibition of REV-ERBβand autophagy as a novel pharmacological approach to induce cytotoxicity in cancer cells[J].Oncogene,2015,34(20):2597-2608.
[19]Umov F D,Rebar E J,Holmes M C,et al.Genome editing with engineered zinc finger nucleases[J].Nat Rev Genet,2010,11(9):636-646.
[20]Li T,Huang S,Zhao X,et al.Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes[J].Nucleic Acids Res,2011,39(14):6315-6325.
[21]Ji Q,Fischer A L,Brown C R,et al.Engineered zinc-finger transcription factors activate OCT4(POU5F1),SOX2,KLF4,c-MYC(MYC)and miR302/367[J].Nucleic Acids Res,2014,42(10):6158-6167.
[22]Sommer D,Peters A,Wirtz T,et al.Efficient genome engineering by targetedhomologous recombination in mouse embryos using transcription activator-like effector nucleases[J/OL].Nat Commun,2014,5:3045.doi:10.1038/ncomms4045.
[23]Cho S W,Kim S,Kim Y,et al.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases[J].Genome Res,2014,24(1):132-141.
[24]Konermann S,Brigham M D,Trevino A E,et al.Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex[J].Nature,2015,517(7536):583-588.
[25]Xiao D,Zhang W,Li Y,et al.A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene[J].J Biotechnol,2015,219:110-116.
[26]Sato S,Sakurai T,Ogasawara J,et al.A circadian clock gene,Rev-erbα,modulates the inflammatory function of macrophages through the negative regulation of Ccl2 expression[J].J Immunol,2014,192(1):407-417.
[27]Nam D,Chatterjee S,Yin H,et al.Novel function of Rev-erbαin promoting brown adipogenesis[J/OL].Sci Rep,2015,5:11239.doi:10.1038/srep11239.
[28]Wang W,Zhang Q,Zhao R,et al.Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2015,31(9):1175-1178.
[29]黄红艳,张彦斌,王小利,等.靶向敲低蛋白1抑制Hep G2肝癌细胞的增殖及迁移[J].细胞与分子免疫学杂志,2015,31(6):782-786.