正选择基因无启动子的供体质粒pN(CDS)(N2)-R的构建及应用
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摘要
为提高对发生基因编辑细胞的筛选效率,本研究构建了具备正负筛选功能的供体质粒pN(CDS)(N2)-R,其正选择基因是无启动子NeoR,负选择基因是能自主表达的RED。利用该质粒和CRISPR-Cas9系统对小鼠B16细胞FasL进行编辑。G418筛选2周,获得混合克隆。镜下观察显示,混合克隆质量较高。junction PCR结果表明,有基因编辑发生。DNA测序结果表明,NeoR准确插入靶位点。细胞杀伤实验表明,FasL功能被有效敲除。该供体质粒的多克隆位点,充分利用同尾酶酶切位点,因而具有广泛的适用性。综上所述,在对各种位点进行编辑时,使用该供体质粒有助于混和克隆的筛选。
To promote the selection efficiency for the gene-edited cells, a positive and negative selection donor plasmid pN(CDS)(N2)-R was constructed, with a promoterless NeoR as the positive selection marker, and a RED with its own enhancer and promoter as the negative selection gene. Combined with the CRISPR/Cas9,the donor plasmid was used to edit the FasL gene of the mouse B16 cells. After G418 selection for two weeks, the pooled clone exhibited a relatively high quality. The result of junction PCR showed that gene editing occurred at the FasL locus, and sequencing results confirmed the accurate insertion of NeoR into the target site. To detect the effect of gene editing, the pooled clone was co-cultured with the T lymphocyte cells and it was more sensitive than normal B16 cells, indicating that the FasL function was knocked out effectively. Because the isocaudarner cleavage site was used as the multiple clone site, so this donor plasmid could be suitable to screen for various loci for gene editing. In summary, this donor plasmid could be conducive to pooled clone selection in gene editing at various target sites.
To promote the selection efficiency for the gene-edited cells, a positive and negative selection donor plasmid pN(CDS)(N2)-R was constructed, with a promoterless NeoR as the positive selection marker, and a RED with its own enhancer and promoter as the negative selection gene. Combined with the CRISPR/Cas9,the donor plasmid was used to edit the FasL gene of the mouse B16 cells. After G418 selection for two weeks, the pooled clone exhibited a relatively high quality. The result of junction PCR showed that gene editing occurred at the FasL locus, and sequencing results confirmed the accurate insertion of NeoR into the target site. To detect the effect of gene editing, the pooled clone was co-cultured with the T lymphocyte cells and it was more sensitive than normal B16 cells, indicating that the FasL function was knocked out effectively. Because the isocaudarner cleavage site was used as the multiple clone site, so this donor plasmid could be suitable to screen for various loci for gene editing. In summary, this donor plasmid could be conducive to pooled clone selection in gene editing at various target sites.
引文
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[2] Garneau J,Dupuis M,Villion M,et al.The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA[J].Nature,2010,468(7320):67-71
[3] 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
[4] Gasiunas G,Barrangou R,Horvath P,et al.Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J].Proc Natl Acad Sci USA,2012,109(39):E2579-2586
[5] Cong L,Ran FA,Cox D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823
[6] Mali P,Yang L,Esvelt K,et al.RNA-guided human genome engineering via Cas9[J].Science,2013,339(6121):823-826
[7] Jinek M,East A,Cheng A,et al.RNA-programmed genome editing in human cells[J].Elife,2013,2:e00471
[8] Cheng AW,Wang H,Yang H,et al.Multiplexed activation of endogenous genes by CRISPR-on,an RNA-guided transcriptional activator system[J].Cell Res,2013,(23):1163-1171
[9] Mali P,Aach J,Stranges PB,et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering[J].Nat Biotechnol,2013,31(9):833-838
[10] Gilbert LA,Larson MH,Morsut L,et al.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J].Cell,2013,154(2):442-451
[11] Qi LS,Larson MH,Gilbert LA,et al.Repurposing CRISPR as an RNA-guided platform for sequence- specific control of gene expression[J].Cell,2013,152(5):1173-1183
[12] Liu XS,Wu H,Ji X,et al.Editing DNA methylation in the mammalian genome[J].Cell,2016,167(1):233-247.e17
[13] San Filippo J,Sung P,Klein H.Mechanism of eukaryotic homologous recombination[J].Annu Rev Biochem,2008,77:229-257
[14] Mao Z,Bozzella M,Seluanov A,et al.Comparison of nonhomologous end joining and homologous recombination in human cells[J].DNA Repair (Amst),2008,7(10):1765-1771
[15] Lieber MR.The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway[J].Annu Rev Biochem,2010,79:181-211
[16] Shalem O,Sanjana NE,Hartenian E,et al.Genome-scale CRISPR-Cas9 knockout screening in human cells[J].Science,2014,343(6166):84-87
[17] Smithies O,Gregg RG,Boggs SS,et al.Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination[J].Nature,1985,317(6034):230-234
[18] Thomas KR,Folger KR,Capecchi MR.High frequency targeting of genes to specific sites in the mammalian genome[J].Cell,1986,44(3):419-428
[19] Chen F,Pruett-Miller SM,Huang Y,et al.High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases[J].Nat Methods,2011,8(9):753-755
[20] Ran FA,Hsu PD,Wright J,et al.Genome engineering using the CRISPR-Cas9 system[J].Nat Protoc,2013,8(11):2281-2308
[21] Mansour SL,Thomas KR,Capecchi MR.Disruption of the proto-oncogene int-2 in mouse embryo- derived stem cells:a general strategy for targeting mutations to non-selectable genes[J].Nature,1988,336 (6197):348-352
[22] Capecchi MR.The new mouse genetics:altering the genome by gene targeting[J].Trends Genet,1989,5(3):70-76
[23] Wu S,Ying G,Wu Q,et al.A protocol for constructing gene targeting vectors:generating knockout mice for the cadherin family and beyond[J].Nat Protoc,2008,3(6):1056-1076
[24] Lee JS,Kallehauge TB,Pedersen LE,et al.Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway[J].Sci Rep,2015,5:8572
[25] Roth TL,Puig-Saus C,Yu R,et al.Reprogramming human T cell function and specificity with non-viral genome targeting[J].Nature,2018,559(7714):405-409
[26] Barzel A,Paulk NK,Shi Y,et al.Promoterless gene targeting without nucleases ameliorates haemophilia B in mice[J].Nature,2015,517(7534):360-364
[27] Li Y,Fan Y,Xu J,et al.Genome-wide RNA-Seq identifies Fas/FasL-mediated tumoricidal activity of embryonic stem cells[J].Int J Cancer,2018,142(9):1829-1841
[28] Su S,Hu B,Shao J,et al.CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients[J].Sci Rep,2016,6:20070
[29] 王英泽,王巧兰,杜朝,等.羧基化单壁碳纳米管对小鼠CIK细胞表型及功能的影响研究[J].中国免疫学杂志(Wang YZ,Wang QL,Du C,et al.Effects on phenotype and function of mouse CIK cells treated with carboxylated single-walled carbon nanotubes[J],Chin J Immunol),2017,33(11):1616-1620
[30] Eyquem J,Mansilla-Soto J,Giavridis T,et al.Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection[J].Nature,2017,543(7643):113-117
[31] Jansen T,Tyler B,Mankowski JL,et al.FasL gene knock-down therapy enhances the antiglioma immune response[J].Neuro Oncol,2010,12(5):482-489附图
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