应用双靶标CRISPR慢病毒载体在间充质干细胞中稳定敲除lncRNA DANCR基因
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摘要
【目的】应用CRISPR/Cas9方法,构建特异性靶向长链非编码RNA DANCR基因两端的双靶标慢病毒载体,稳定敲除间充质干细胞(MSC)中的DANCR基因,为研究DANCR的生物学功能奠定基础。【方法】首先设计靶向DANCR的5’和3’端的sgRNA(single-guide RNA),转染293FT细胞,提取293FT细胞基因组DNA,验证靶向序列的有效性。然后通过gateway和酶切连接的方法,将分别靶向5’和3’端的两条有效sgRNA序列连接至同一慢病毒CRISPR载体。最终使用293FT进行慢病毒包装,收获病毒感染MSC,检测MSC中DANCR敲除效率。【结果】在证明靶向DANCR基因两端的sgRNA序列均单独有效的基础上,将靶向5’和3’端的sgRNA进行组合,成功构建靶向DANCR的双靶标慢病毒载体。将载体进行慢病毒包装后感染MSC,成功获得DANCR敲除的MSC。【结论】应用CRISPR方法成功构建敲除DANCR的慢病毒载体,能高效稳定地敲除MSC中的DANCR基因。
【Objective】Using the CRISPR/Cas9(CRISPR/crispr-associated(Cas)9 method,a dual-target lentiviral vector containing single-guide RNAs(sgRNAs)targeting both the 5'and 3'ends of the anti-differentiation noncoding RNA(DANCR)gene was constructed. Stable knockout of DANCR gene in mesenchymal stem cells(MSC)would be help?ful for the future study of the biological function of DANCR.【Methods】Designed sgRNAs targeting either the 5'or 3'end of DANCR and cloned into two CRISPR vectors. The vector was transfected into 293 FT cells,and the genomic DNA of 293 FT cells was extracted to verify the efficiency of individual sequence. Two functional sgRNAs targeting either the 5'or 3'end were incorporated into a same lentiviral CRISPR vector through gateway and enzymatic ligation. 293 FT was used for lentiviral packaging,after which the virus was harvested to infect MSC,and the knockout efficiency of DANCR in MSC was detected.【Results】All four sgRNA sequences targeting DANCR successfully guided Cas9 to cleave the gene.sgRNAs targeting either the 5'and 3'end were combined to establish a dual-target lentiviral vector for stable knockout of DANCR. The vector was packaged into lentivirus and infected MSC. Finally,we successfully obtained mesenchymal stem cell lines with DANCR gene knockout.【Conclusions】Using the CRISPR method,a dual-target lentiviral vector can effi?ciently and stably knock out DANCR gene in MSC.
【Objective】Using the CRISPR/Cas9(CRISPR/crispr-associated(Cas)9 method,a dual-target lentiviral vector containing single-guide RNAs(sgRNAs)targeting both the 5'and 3'ends of the anti-differentiation noncoding RNA(DANCR)gene was constructed. Stable knockout of DANCR gene in mesenchymal stem cells(MSC)would be help?ful for the future study of the biological function of DANCR.【Methods】Designed sgRNAs targeting either the 5'or 3'end of DANCR and cloned into two CRISPR vectors. The vector was transfected into 293 FT cells,and the genomic DNA of 293 FT cells was extracted to verify the efficiency of individual sequence. Two functional sgRNAs targeting either the 5'or 3'end were incorporated into a same lentiviral CRISPR vector through gateway and enzymatic ligation. 293 FT was used for lentiviral packaging,after which the virus was harvested to infect MSC,and the knockout efficiency of DANCR in MSC was detected.【Results】All four sgRNA sequences targeting DANCR successfully guided Cas9 to cleave the gene.sgRNAs targeting either the 5'and 3'end were combined to establish a dual-target lentiviral vector for stable knockout of DANCR. The vector was packaged into lentivirus and infected MSC. Finally,we successfully obtained mesenchymal stem cell lines with DANCR gene knockout.【Conclusions】Using the CRISPR method,a dual-target lentiviral vector can effi?ciently and stably knock out DANCR gene in MSC.
引文
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[10]Lu Y,Hu Z,Mangala LS,et al. MYC TargetedLong Noncoding RNA DANCR Promotes Cancer inPart by Reducing p21 Levels[J]. Cancer Res,2018,78(1):64-74.
[11]Ma Y,Zhou G,Li M,et al. Long noncoding RNADANCR mediates cisplatin resistance in gliomacells via activating AXL/PI3K/Akt/NF-kappaBsignaling pathway[J]. Neurochem Int,2018,118:233-241.
[12]Jia Q,Chen X,Jiang W,et al. The RegulatoryEffects of Long Noncoding RNA-ANCR on DentalTissue-Derived Stem Cells[J]. Stem Cells Int,2016,2016:3146805.
[13]Zhang L,Sun X,Chen S,et al. Long noncodingRNA DANCR regulates miR-1305-Smad 4 axis topromote chondrogenic differentiation of humansynovium-derived mesenchymal stem cells[J].Biosci Rep,2017,37(4),pii:BSR20170347.
[14]Zhang L,Yang C,Chen S,et al. Long NoncodingRNA DANCR Is a Positive Regulator of Proliferationand Chondrogenic Differentiation in HumanSynovium-Derived Stem Cells[J]. DNA Cell Biol,2017,36(2):136-142.
[15]Fan X,Yuan J,Xie J,et al. Long non-proteincoding RNA DANCR functions as a competingendogenous RNA to regulate osteoarthritisprogression via miR-577/SphK2 axis[J]. BiochemBiophys Res Commun,2018,500(3):658-664.
[2] Wang Y,Zeng X,Wang N,et al. Long noncodingRNA DANCR, working as a competitiveendogenous RNA, promotes ROCK1-mediatedproliferation and metastasis via decoying of miR-335-5p and miR-1972 in osteosarcoma[J]. MolCancer,2018,17(1):89.
[3] Tang Z,Gong Z,Sun X,et al. LncRNA DANCRinvolved osteolysis after total hip arthroplasty byregulating FOXO1 expression to inhibit osteoblastdifferentiation[J]. J Biomed Sci,2018,25(1):4.
[4] Zhang L,Chen S,Bao N,et al. Sox4 enhanceschondrogenic differentiation and proliferation ofhuman synovium-derived stem cell via activation oflong noncoding RNA DANCR[J]. J Mol Histol,2015,46(6):467-473.
[5] Zhang J,Tao Z,Wang Y. Long noncoding RNADANCR regulates the proliferation and osteogenicdifferentiation of human bone-derived marrowmesenchymal stem cells via the p38 MAPK pathway[J]. Int J Mol Med,2018,41(1):213-219.
[6] Ran FA, Hsu PD, Wright J, et al. Genomeengineering using the CRISPR-Cas9 system[J].Nature Protocols,2013,8(11):2281-2308.
[7] Yao X, Wang X, Hu XD, et al. Homology-mediated end joining-based targeted integrationusing CRISPR/Cas9[J]. Cell Research,2017,27(6):801-814.
[8] Cong L,Ran FA,Cox D,et al. Multiplex genomeengineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823.
[9] Yuan SX,Wang J,Yang F,et al. Long noncodingRNA DANCR increases stemness features ofhepatocellular carcinoma by derepression ofCTNNB1[J]. Hepatology,2016,63(2):499-511.
[10]Lu Y,Hu Z,Mangala LS,et al. MYC TargetedLong Noncoding RNA DANCR Promotes Cancer inPart by Reducing p21 Levels[J]. Cancer Res,2018,78(1):64-74.
[11]Ma Y,Zhou G,Li M,et al. Long noncoding RNADANCR mediates cisplatin resistance in gliomacells via activating AXL/PI3K/Akt/NF-kappaBsignaling pathway[J]. Neurochem Int,2018,118:233-241.
[12]Jia Q,Chen X,Jiang W,et al. The RegulatoryEffects of Long Noncoding RNA-ANCR on DentalTissue-Derived Stem Cells[J]. Stem Cells Int,2016,2016:3146805.
[13]Zhang L,Sun X,Chen S,et al. Long noncodingRNA DANCR regulates miR-1305-Smad 4 axis topromote chondrogenic differentiation of humansynovium-derived mesenchymal stem cells[J].Biosci Rep,2017,37(4),pii:BSR20170347.
[14]Zhang L,Yang C,Chen S,et al. Long NoncodingRNA DANCR Is a Positive Regulator of Proliferationand Chondrogenic Differentiation in HumanSynovium-Derived Stem Cells[J]. DNA Cell Biol,2017,36(2):136-142.
[15]Fan X,Yuan J,Xie J,et al. Long non-proteincoding RNA DANCR functions as a competingendogenous RNA to regulate osteoarthritisprogression via miR-577/SphK2 axis[J]. BiochemBiophys Res Commun,2018,500(3):658-664.
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