水牛asf1a基因克隆及高效敲除靶位点的筛选
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摘要
本研究旨在对水牛anti-silence factor 1a (asf1a)基因进行克隆并行生物信息学分析,并进一步筛选基于CRISPR/Cas9基因编辑系统的高效敲除asf1a基因位点。根据NCBI公布的牛asf1a基因mRNA序列设计特异性引物,以水牛GV期卵母细胞RNA反转录后的cDNA作为模板,通过PCR技术克隆获得asf1a基因序列片段,利用生物信息学分析方法,对克隆得到的序列进行和相应翻译的蛋白质进行了分析。在克隆得到的水牛asf1a基因序列上筛选3个PAM位点,并根据PAM位点构建3个gRNA载体(g1、g2、g3)以及相应的RGS载体,将pcDNA3.1-h Cas9连同gRNA、RGS 3种质粒按照3∶1∶1比例共转293T细胞,筛选高效的CRISPR/Cas9敲除位点。试验以水牛GV期的卵母细胞RNA作为模板,通过RT-PCR技术克隆获得asf1a基因序列,并据测序结果设计靶向区域筛选高效asf1a基因敲除位点。结果发现,水牛asf1a基因CDS区全长615 bp,编码204个氨基酸;多重序列分析显示,水牛asf1a基因核苷酸序列与黄牛、绵羊、猪、人、小鼠同源性分别达到了99%,98%,97%,96%,93%;蛋白质结构预测分析发现存在ASF1-hist-chap等结构,二级结构含有9个α螺旋、12个β折叠、14个T转角、14个无规则卷曲。系统进化树分析表明,水牛和黄牛亲缘性最接近。根据水牛asf1a设计的敲除载体转染293T和成纤维细胞后,细胞基因组进行T7E1酶切的结果表明,g1位点对水牛成纤维细胞敲除效率最高,达到93.82%,同时,sanger测序表明效率达17/20。本研究成功克隆了水牛asf1a基因和分析了其生物学信息,同时筛选出了asf1a的高效敲除位点,为研究水牛asf1a基因功能奠定基础。
This paper aimed to cloning buffalo anti-silence factor 1 a(asf1a) gene,bioinformatics analysis,and using the CRISPR/CAS9 gene editing system to screen out the sites of efficient knockout.Specific primers were designed according to the NCBI published asf1a mRNA sequence and asf1a gene were amplified and cloned by RT-PCR.The nucleotide sequnce and protein structure of CDS region were analyzed through bioinformatic method.Three gRNA vectors(g1,g2,g3) and the corresponding RGS vectors were constructed according to the PAM sites.The pcDNA3.1-h Cas9,together with gRNA,RGS were co-transfected into 293 T cells in a ratio of 3∶1∶1,and highly efficient CRISPR/Cas9 knockout sites were screened.The results showed that the asf1a gene CDS region was 615 bp and encoded 204 amino acids by RT-PCR technique,using the oocyte RNA of the buffalo GV period.Multiple sequence analysis showed that the nucleotide sequences of the buffalo asf1a gene were homologous to cattle,sheep,pigs,human and mice,reaching 99%,98%,97%,96%,93%respectively.Prediction of protein structure found that there were asf1-hist-chap structure,secondary structure contains 9α-helix,12β-sheet,14 T-angle,14 random curls.The results of T7 E1 enzyme-cut showed that the g1 of buffalo fibroblasts was the highest,reaching 93.82%,at the same time,the Sanger sequencing showed an efficiency of 17/20.This study successfully cloned buffalo asf1 agene and screened out the effective knockout sites,which lay a foundation for studying asf1 agene functions.
This paper aimed to cloning buffalo anti-silence factor 1 a(asf1a) gene,bioinformatics analysis,and using the CRISPR/CAS9 gene editing system to screen out the sites of efficient knockout.Specific primers were designed according to the NCBI published asf1a mRNA sequence and asf1a gene were amplified and cloned by RT-PCR.The nucleotide sequnce and protein structure of CDS region were analyzed through bioinformatic method.Three gRNA vectors(g1,g2,g3) and the corresponding RGS vectors were constructed according to the PAM sites.The pcDNA3.1-h Cas9,together with gRNA,RGS were co-transfected into 293 T cells in a ratio of 3∶1∶1,and highly efficient CRISPR/Cas9 knockout sites were screened.The results showed that the asf1a gene CDS region was 615 bp and encoded 204 amino acids by RT-PCR technique,using the oocyte RNA of the buffalo GV period.Multiple sequence analysis showed that the nucleotide sequences of the buffalo asf1a gene were homologous to cattle,sheep,pigs,human and mice,reaching 99%,98%,97%,96%,93%respectively.Prediction of protein structure found that there were asf1-hist-chap structure,secondary structure contains 9α-helix,12β-sheet,14 T-angle,14 random curls.The results of T7 E1 enzyme-cut showed that the g1 of buffalo fibroblasts was the highest,reaching 93.82%,at the same time,the Sanger sequencing showed an efficiency of 17/20.This study successfully cloned buffalo asf1 agene and screened out the effective knockout sites,which lay a foundation for studying asf1 agene functions.
引文
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[29] LEE K Y,IM J S,SHIBATA E,et al.ASF1a Promotes non-homologous end joining repair by facilitating phosphorylation of MDC1 by ATM at double-strand breaks[J].Mol Cell,2017,68:61-75.
[30] RAMEY C J,HOWAR S,ADKINS M,et al.Activation of the DNA damage checkpoint in yeast lacking the histone chaperone anti-silencing function 1[J].Mol Cell Biol,2004,24:10313-10327.
[31] TSABAR M,WATERMAN D P,AGUILAR F,et al.Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair[J].Genes Dev,2016,30:1211-1224.
[32] KIM J AHABER J E.Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete[J].Proc Natl Acad Sci U S A,2009,106:1151-1156.
[2] MOUSSON F,LAUTRETTE A,THURET J Y,et al.Structural basis for the interaction of Asf1 with histone H3 and its functional implications[J].Proc Natl Acad Sci U S A,2005,102:5975-80.
[3] ENGLISH C M,ADKINS M W,CARSON J J,et al.Structural basis for the histone chaperone activity of Asf1[J].Cell,2006,127:495-508.
[4] BATTU A,RAY A,WANI A A.ASF1A and ATM regulate H3K56-mediated cell-cycle checkpoint recovery in response to UV irradiation[J].Nucleic Acids Res,2011,39:7931-7945.
[5] TYLER J K,ADAMS C R,CHEN S R,et al.The RCAF complex mediates chromatin assembly during DNA replication and repair[J].Nature,1999,402:555-560.
[6] PRADO F,CORTES-LEDESMA F,AGUILERA A.The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange[J].EMBO Rep,2004,5:497-502.
[7] BETTS D H,PERRAULT S D,PETRIK J,et al.Telomere length analysis in goat clones and their offspring[J].Mol Reprod Dev,2005,72:461-470.
[8] LOYOLA AALMOUZNI G.Histone chaperones,a supporting role in the limelight[J].Biochim Biophys Acta,2004,1677(1/3):3-11.
[9] TANG Y,POUSTOVOITOV M V,ZHAO K,et al.Structure of a human ASF1a-HIRA complex and insights into specificity of histone chaperone complex assembly[J].Nat Struct Mol Biol,2006,13:921-929.
[10] SHARP J A,FOUTS E T,KRAWITZ D C,et al.Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing[J].Curr Biol,2001,11:463-473.
[11] TYLER J K,ADAMS C R,CHEN S R,et al.The RCAF complex mediates chromatin assembly during DNA replication and repair[J].Nature,1999,402:555-560.
[12] ZHANG R,POUSTOVOITOV M V,YE X,et al.Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA[J].Dev Cell,2005,8:19-30.
[13] KIROV N,SHTILBANS A,RUSHLOW C.Isolation and characterization of a new gene encoding a member of the HIRA family of proteins from Drosophila melanogaster[J].Gene,1998,212:323-332.
[14] TANG Y,POUSTOVOITOV M V,ZHAO K,et al.Structure of a human ASF1a-HIRA complex and insights into specificity of histone chaperone complex assembly[J].Nat Struct Mol Biol,2006,13:921-929.
[15] WILMUT I,SCHNIEKE A,MCWHIR J,et al.Viable offspring derived from fetal and adult mammalian cells[J].Nature,1997,385:810-813.
[16] TAKAHASHI KYAMANAKA S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J].Cell,2006,126:663-676.
[17] TAKAHASHI K,TANABE K,OHNUKI M,et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J].Cell,2007,131:861-872.
[18] YU J,VODYANIK M A,SMUGAOTTO K,et al.Induced pluripotent stem cell lines derived from human somatic cells[J].Science,2007,318:1917-1920.
[19] CAO H,YANG P,PU Y,et al.Characterization of bovine induced pluripotent stem cells by lentiviral transduction of reprogramming factor fusion proteins[J].Int J Biol Sci,2012,8:498-511.
[20] DENG Y,LIU Q,LUO C,et al.Generation of induced pluripotent stem cells from buffalo (Bubalus bubalis) fetal fibroblasts with buffalo defined factors[J].Stem Cell Deve,2012,21:2485-2494.
[21] SHI H,FU Q,LI G,et al.Roles of p53 and ASF1A in the reprogramming of sheep kidney cells to pluripotent cells[J].Cell Reprogram,2015,17:441-452.
[22] MALI P,YANG L,ESVELT K M,et al.RNA-guided human genome engineering via Cas9[J].Science,2013,339:823-826.
[23] CONG L,RAN F A,COX D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339:819-823.
[24] PEREZPINERA P,KOCAK D D,VOCKLEY C M,et al.RNA-guided gene activation by CRISPR-Cas9-based transcription factors[J].Nat Methods,2013,10:973-976.
[25] HSU P D,LANDER E S,ZHANG F.Development and applications of CRISPR-Cas9 for genome engineering[J].Cell,2014,157:1262-1278.
[26] DONHAM D C,SCORGIE J K,CHURCHILL M E A.The activity of the histone chaperone yeast Asf1 in the assembly and disassembly of histone H3/H4-DNA complexes[J].Nucleic Acids Res,2011,39:5449-5458.
[27] SCHWABISH M ASTRUHL K.Asf1 mediates histone eviction and deposition during elongation by RNA polymerase II[J].Mol Cell,2006,22:415-422.
[28] ADKINS M W,HOWAR S R,TYLER J K.Chromatin disassembly mediated by the histone chaperone Asf1 is essential for transcriptional activation of the yeast PHO5 and PHO8 genes[J].Mol Cell,2004,14(5):657-666.
[29] LEE K Y,IM J S,SHIBATA E,et al.ASF1a Promotes non-homologous end joining repair by facilitating phosphorylation of MDC1 by ATM at double-strand breaks[J].Mol Cell,2017,68:61-75.
[30] RAMEY C J,HOWAR S,ADKINS M,et al.Activation of the DNA damage checkpoint in yeast lacking the histone chaperone anti-silencing function 1[J].Mol Cell Biol,2004,24:10313-10327.
[31] TSABAR M,WATERMAN D P,AGUILAR F,et al.Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair[J].Genes Dev,2016,30:1211-1224.
[32] KIM J AHABER J E.Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete[J].Proc Natl Acad Sci U S A,2009,106:1151-1156.