CN108913692B - 特异性靶向SATB1基因的sgRNA及其在转录激活中的应用 - Google Patents
特异性靶向SATB1基因的sgRNA及其在转录激活中的应用 Download PDFInfo
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Abstract
本发明提供一种制备特异性靶向目标基因SATB1的sgRNA，序列为SEQ ID NO.1，本发明利用Casilio系统特异性转录激活人Hela细胞中SATB1基因。本发明制备的特异性靶向人SATB1基因的sgRNA能够精确靶向人SATB1基因并且实现转录激活。本发明制备方法步骤简单、sgRNA靶向性好，SATB1转录激活效率高。本发明可以在较短时间内实现SATB1转录激活，与直接细胞内转染SATB1过表达质粒相比，更符合生理情况，且价格低廉，有助于进一步深入研究SATB1的生物学功能。
Description
技术领域
本发明属于基因工程领域，更具体地说涉及一种特异性靶向SATB1基因的sgRNA，以及利用该sgRNA通过Casilio系统特异性转录激活人Hela细胞SATB1基因的方法。
背景技术
规律成簇间隔短回文重复系统(clustered regularly interspaced shortpalindromic repeat；CRISPR-associated，CRISPR-Cas9)是一种具有核酸内切酶活性的复合体，识别特定的DNA序列，进行特定位点切割造成双链DNA断裂(Double-strand breaks,DSB)。而核酸酶缺陷突变体dCas9则去除了Cas9的切割能力，仅保留了sgRNA介导的靶向能力。将转录激活因子P65HSF1及RNA结合蛋白Pumilio/FBF(PUF)融合表达，同时，在sgRNA后串联PUF结合位点，并与dCas9共同表达，可以利用sgRNA和dCas9将PUF结合位点靶向至目的基因，利用PUF与PUF结合位点结合，将转录激活因子P65HSF1靶向至目的基因，从而转录激活该基因。此系统称为Casilio系统(见图1)。
dCas9靶向DNA是通过向导核糖核酸(guide RNA，sgRNA)和靶序列互补识别的原理实现的。sgRNA的特异性决定了基因编辑的精确程度。因此，设计、制备出精确性和特异性靶向目标基因的sgRNA成为CRISPR-Cas9基因敲除的关键技术。
SATB1是一类转录因子，具有转录目的基因等多个生物学功能。利用Casilio系统快速、简便、高效、特异性转录激活SATB1基因，有助于深入研究其功能。而能否设计、制备出精确性和特异性靶向SATB1基因的sgRNA以及通过分子生物学方法制备出靶向SATB1基因的Casilio系统成为实现SATB1基因激活的关键技术。
参考文献：
Ran,F.A.et al.,Genome engineering using the CRISPR-Cas9system.NATPROTOC 8 2281 (2013)。
Cheng,A.W.et al.,Casilio:a versatile CRISPR-Cas9-Pumilio hybrid forgene regulation and genomic labeling.CELL RES 26 254(2016).
发明内容
本发明的目的是提供一种制备特异性靶向目标基因SATB1的sgRNA，序列为：ttgcggaagaagcagatgtcagg(SEQ ID NO.1)。
本发明的另一个目的是提供所述sgRNA在转录激活靶向SATB1基因中的应用，是基于 Casilio系统特异性转录激活人Hela细胞SATB1基因，通过以下步骤实现。
1.靶向SATB1基因sgRNA寡核苷酸的设计和选择：
(1)在SATB1基因上选择特征为5’-N(19)GG或5’-N(20)GG-3’或5’-N(21)GG-3’的序列；
(2)sgRNA在SATB1基因上的靶向位点位于转录起始位点后200bp内；
按以上原则，设计得到sgRNA，序列为SEQ No.1。
2.利用sgRNA，合成可以连入pAC-sgRNA-5×PBSa真核表达载体的双链sgRNA寡聚核苷酸：
(1)根据选择的sgRNA，去除3’端NGG三个碱基，在其5’端加上CACCG得到正向寡核苷酸；根据选择的sgRNA，去除3’端NGG三个碱基，获得对应DNA的互补链，并且在其5’加上AAAC,3’端加上C得到反向寡核苷酸。上述正向、反向寡核苷酸序列见SEQ NO.2和SEQ NO.3；
(2)分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的sgRNA寡聚核苷酸变性、退火，合成可以连入pAC-sgRNA-5×PBSa真核表达载体的双链。
3.将sgRNA构建至pAC-sgRNA-5×PBSa载体，鉴定并扩增：
(1)将退火的sgRNA寡聚核苷酸双链与pAC-sgRNA-5×PBSa载体(序列见SEQ NO.4)连接获得pAC-SATB1sgRNA-5×PBSa质粒(序列见SEQ NO.5)；
(2)pAC-SATB1sgRNA-5×PBSa质粒转化感受态细菌并涂Amp+平板；
(3)对pAC-SATB1sgRNA-5×PBSa质粒进行鉴定：挑选阳性克隆并用序列如SEQ IDNO.6 所示的通用引物U6测序的方法鉴定阳性克隆，37℃摇床摇阳性克隆菌过夜并用抽提pAC-SATB1sgRNA-5×PBSa质粒。
4.转染Hela细胞转录激活SATB1：
(1)按照DNA&siRNA Transfection Reagent(Polyplus transfection，114-01)的操作手册，用800ng脂质体装载的pAC-SATB1sgRNA-5×PBSa质粒、400ng 的pmax-dCas9质粒((序列见SEQ No.7))以及800ng的pAC1393-PUFa-P65-HSF1质粒(序列见SEQNo.8)，分别转染至Hela细胞；
(2)转染48小时后收集转然后的Hela细胞，提取总RNA和总蛋白，Real time Q-PCR及Western Blot确认SATB1被转录激活。
本发明可以在较短时间内实现SATB1转录激活，与直接细胞内转染SATB1过表达质粒相比，更符合生理情况，且价格低廉，有助于进一步深入研究SATB1的生物学功能。利用本发明制备的特异性靶向人SATB1基因的sgRNA能够精确靶向人SATB1基因并且实现SATB1转录激活。该制备方法步骤简单、sgRNA靶向性好，SATB1基因转录激活效率高。
附图说明
图1是本发明的Casilio系统原理图。
图2是SATB1mRNA表达情况的分析图，其中示出通过Real time Q-PCR确认SATB1mRNA 上调。
图3是用Western Blot检测SATB1表达水平，确认转录激活后的SATB1蛋白水平上调。
具体实施方式
下面结合附图和具体的实施例对本发明的技术方案做进一步介绍。
实施例1靶向人SATB1基因的sgRNA的设计和合成
1.靶向人SATB1基因的sgRNA的设计。
(1)在SATB1基因上选择特征为5’-N(19)GG或5’-N(20)GG-3’或5’-N(21)GG-3’的序列。
(2)sgRNA在SATB1基因上的靶向位点位于转录起始位点后200bp内。
2.按以上原则，设计得到sgRNA，序列为SEQ No.1。
3.靶向人SATB1基因的sgRNA寡聚核苷酸的合成和构建。
根据选择的sgRNA，去除3’端NGG三个碱基，在其5’端加上CACCG得到正向寡核苷酸；根据选择的sgRNA，去除3’端NGG三个碱基，获得对应DNA的互补链，并且在其5’加上AAAC,3’端加上C得到反向寡核苷酸。序列见SEQ NO.2和SEQ NO.3。分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的sgRNA寡聚核苷酸变性、退火，形成可以连入 pAC-sgRNA-5×PBSa真核表达载体的双链sgRNA寡聚核苷酸。
变性、退火反应体系为：
1μl正向寡核苷酸(100μM)
1μl反向寡核苷酸(100μM)
1μl T4ligation buffer(New England BioLabs公司，货号：B0202S)
1μl T4PNK(New England BioLabs公司，货号：M0201S)
6μl灭菌水。
在PCR仪中按照以下程序运行：37℃，30min；95℃,5min；0.1℃/sec由95℃降至25℃。
实施例2将双链sgRNA构建至pAC-sgRNA-5×PBSa载体
将变性、退火后所得双链sgRNA按1：200比例稀释。
将双链sgRNA构建至pAC-sgRNA-5×PBSa载体(序列见SEQ NO.4)，反应体系如下：
100ng pAC-sgRNA-5×PBSa质粒
2μl稀释后双链sgRNA
2μl Tango buffer，10X(Thermo Scientific,货号BY5)
1μl DTT，10mM(Thermo Scientific,货号R0862)
1μl ATP，10mM(Thermo Scientific,货号AM8110G)
1μl FastDigest BbsⅠ(Thermo Scientific,货号FD1014)
0.5μl T7ligase(Enzymatics,货号L602L)
灭菌水，补足至20μl。
反应条件：37℃5min，21℃5min。共6个循环.
将上述步骤获得的连接产物转化Stbl3感受态细胞(Thermo Scientific,货号C7373-03) 并涂Amp+平板(50μg/ml)，并挑取克隆。
用如序列表SEQ ID NO.6所示的通用引物U6，用Sanger法测序的方法鉴定获得阳性克隆。 37℃摇床摇菌过夜培养阳性克隆，抽提质粒，获得pAC-SATB1sgRNA-5×PBSa质粒(如序列表SEQ ID NO.5所示)。
实施例3构建人Hela细胞SATB1基因转录激活
1、细胞培养与转染。
(1)Hela细胞接种培养于DMEM高糖培养液中(Thermo Scientific,货号11995-065)，其中含10％FBS(Thermo Scientific,货号10099-141)，penicillin(100U/ml)和streptomycin (100μg/ml)。
(2)在转染前将Hela细胞接种至6孔板中，待70％～80％密度时进行转染。
(3)按照DNA&siRNA Transfection Reagent(Polyplus transfection，114-01) 操作手册，将400ng pmax-dCas9质粒(序列见SEQ No.7),800ng pAC1393-PUFa-P65-HSF1 质粒(序列见SEQ No.8)和800ng pAC-SATB1sgRNA-5×PBSa质粒转染至每孔细胞中，6～8 小时后换液。
2、Real time Q-PCR鉴定SATB1mRNA表达情况：
转染48小时后，收集转染后的Hela细胞，提取总RNA，随后逆转录为cDNA，再进行PCR。反应体系如下：
10μl SYBRPremix Ex Taq(Tli RNaseH Plus)(2X)(TaKaRa，货号RR420A)
0.4μl Forward Primer
0.4μl Reverse Primer(SATB1正反向引物序列见SEQ NO.9和SEQ NO.10；Betaactin 正反向引物序列见SEQ NO.11和SEQ NO.12)
0.4μl ROX Reference DyeⅡ(50X)(TaKaRa，货号RR420A)
50ng cDNA模板
灭菌水(补足至20μl)
反应条件为
Step Ⅰ(1个循环)
95℃30s
Step Ⅱ(40个循环)
95℃5s
60℃34s(此步收集荧光)
根据引物和内参CT值，分析SATB1mRNA表达情况(见图2)
3、Western Blot鉴定SATB1敲除情况：
提取对照组和敲除组细胞蛋白，进行蛋白电泳，随后转膜，封闭，加入抗SATB1一抗孵育(Abcam，货号ab92307)过夜，次日去除一抗并洗涤30min，加入二抗(Abcam，ab6721) 孵育1小时，随后再次洗涤30min后显影，结果见图3。
序列表
<110> 浙江大学
<120> 特异性靶向SATB1基因的sgRNA及其在转录激活中的应用
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Unknown)
<400> 1
ttgcggaaga agcagatgtc agg 23
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Unknown)
<400> 2
caccgttgcg gaagaagcag atgtc 25
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Unknown)
<400> 3
cgacatctgc ttcttccgca acaaa 25
<210> 4
<211> 3270
<212> DNA
<213> 人工序列(Unknown)
<400> 4
ggcgcgccga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg 60
ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt 120
gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg 180
actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt 240
ggaaaggacg aaacaccggg tcttcgagaa gacctgttta agagctatgc tggaaacagc 300
atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 360
ccaattgggt ctccagattg tatgtagcct gtatgtagcc tgtatgtagc ctgtatgtag 420
cctgtatgta agatcttttt ttgttttaga gctagaaata gcaagttaaa ataaggctag 480
tccgtagcgc gtgcgccaat tctgcagaca aatggcggcg cgccgcggcc gcaggaaccc 540
ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 600
ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 660
agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 720
caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 780
gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 840
tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 900
gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 960
atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 1020
cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 1080
ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 1140
aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 1200
ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 1260
acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 1320
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 1380
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 1440
taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 1500
gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 1560
tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 1620
ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 1680
taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 1740
gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 1800
aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 1860
gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 1920
ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 1980
ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 2040
acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 2100
taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 2160
tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 2220
cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 2280
ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg 2340
gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 2400
gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 2460
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 2520
aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 2580
actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 2640
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 2700
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 2760
atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 2820
ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 2880
gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 2940
cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 3000
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 3060
cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 3120
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 3180
gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 3240
tggccttttg ctggcctttt gctcacatgt 3270
<210> 5
<211> 3273
<212> DNA
<213> 人工序列(Unknown)
<400> 5
ggcgcgccga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg 60
ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt 120
gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg 180
actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt 240
ggaaaggacg aaacaccgtt gcggaagaag cagatgtcgt ttaagagcta tgctggaaac 300
agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg 360
gtgccaattg ggtctccaga ttgtatgtag cctgtatgta gcctgtatgt agcctgtatg 420
tagcctgtat gtaagatctt tttttgtttt agagctagaa atagcaagtt aaaataaggc 480
tagtccgtag cgcgtgcgcc aattctgcag acaaatggcg gcgcgccgcg gccgcaggaa 540
cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg 600
cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg 660
cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 720
tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg 780
cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 840
ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 900
atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 960
ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 1020
tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 1080
accctatctc gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt 1140
taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta 1200
caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 1260
gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 1320
acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 1380
cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 1440
taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 1500
tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 1560
aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 1620
ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 1680
aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 1740
acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 1800
ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 1860
gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 1920
atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 1980
acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 2040
tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 2100
ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 2160
aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 2220
aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 2280
ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag 2340
atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 2400
aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 2460
accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 2520
tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 2580
tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 2640
tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 2700
cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 2760
caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 2820
cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 2880
cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 2940
gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 3000
acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 3060
atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 3120
cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 3180
gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 3240
tcctggcctt ttgctggcct tttgctcaca tgt 3273
<210> 6
<211> 24
<212> DNA
<213> 人工序列(Unknown)
<400> 6
atggactatc atatgcttac cgta 24
<210> 7
<211> 8330
<212> DNA
<213> 人工序列(Unknown)
<400> 7
tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60
ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120
aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180
gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240
gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300
agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360
ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420
cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480
gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660
cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg 720
tcgtttagtg aaccgtcaga tcactagtag ctttattgcg gtagtttatc acagttaaat 780
tgctaacgca gtcagtgctc gactgatcac aggtaagtat caaggttaca agacaggttt 840
aaggaggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg 900
cacctattgg tcttactgac atccactttg cctttctctc cacagggaaa aaacaattga 960
caagtttgta caaaaaagca ggctccgaat tcaccggtgc cgccaccatg atcgatggtg 1020
gcggtggtag cgggggaggc ggctccgggg gcggaggcag tatgtaccca tacgatgttc 1080
cagattacgc ttcgccgaag aaaaagcgca aggtcgaagc gtccgacaag aagtacagca 1140
tcggcctggc catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg 1200
tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc 1260
tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa 1320
ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca 1380
gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg 1440
tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg 1500
cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg 1560
acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc 1620
acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc 1680
agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg 1740
acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg 1800
cccagctgcc cggcgagaag aagaatggcc tgttcggcaa cctgattgcc ctgagcctgg 1860
gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga 1920
gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg 1980
ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga 2040
gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg 2100
agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt 2160
acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag 2220
ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg 2280
aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca 2340
acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg 2400
aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc 2460
gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca 2520
gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt 2580
ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg 2640
tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag 2700
tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg 2760
ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg 2820
actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt 2880
tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc 2940
tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg 3000
aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag 3060
tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga 3120
tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg 3180
gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg 3240
acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc 3300
tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc 3360
tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga 3420
accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg 3480
gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc 3540
agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg 3600
aactggacat caaccggctg tccgactacg atgtggacgc catcgtgcct cagagctttc 3660
tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga 3720
gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc 3780
tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg 3840
gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga 3900
tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg 3960
acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc 4020
ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg 4080
cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg 4140
agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc 4200
aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca 4260
agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg 4320
gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc 4380
tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca 4440
aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg 4500
accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg 4560
ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca 4620
ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct 4680
acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg 4740
aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg 4800
ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg 4860
gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg 4920
acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc 4980
tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg 5040
agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact 5100
ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc 5160
tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag 5220
gcgacagccc caagaagaag agaaaggtgg aggccagcgg aggcggcggt agcggaggag 5280
gcgggtccgg cggcggcggt agtgggccgg ccggaggagg tggaagcgga ggaggaggaa 5340
gcggaggagg aggtagcgga cctaagaaaa agaggaaggt ggcggccgct ggatcccctt 5400
cagggcagat cagcaaccag gccctggctc tggcccctag ctccgctcca gtgctggccc 5460
agactatggt gccctctagt gctatggtgc ctctggccca gccacctgct ccagcccctg 5520
tgctgacccc aggaccaccc cagtcactga gcgctccagt gcccaagtct acacaggccg 5580
gcgaggggac tctgagtgaa gctctgctgc acctgcagtt cgacgctgat gaggacctgg 5640
gagctctgct ggggaacagc accgatcccg gagtgttcac agatctggcc tccgtggaca 5700
actctgagtt tcagcagctg ctgaatcagg gcgtgtccat gtctcatagt acagccgaac 5760
caatgctgat ggagtacccc gaagccatta cccggctggt gaccggcagc cagcggcccc 5820
ccgaccccgc tccaactccc ctgggaacca gcggcctgcc taatgggctg tccggagatg 5880
aagacttctc aagcatcgct gatatggact ttagtgccct gctgtcacag atttcctcta 5940
gtgggcaggg aggaggtgga agcggcttca gcgtggacac cagtgccctg ctggacctgt 6000
tcagcccctc ggtgaccgtg cccgacatga gcctgcctga ccttgacagc agcctggcca 6060
gtatccaaga gctcctgtct ccccaggagc cccccaggcc tcccgaggca gagaacagca 6120
gcccggattc agggaagcag ctggtgcact acacagcgca gccgctgttc ctgctggacc 6180
ccggctccgt ggacaccggg agcaacgacc tgccggtgct gtttgagctg ggagagggct 6240
cctacttctc cgaaggggac ggcttcgccg aggaccccac catctccctg ctgacaggct 6300
cggagcctcc caaagccaag gaccccactg tctccatcga ttgattaatt aagaattcga 6360
cccagctttc ttgtacaaag tggttgatat ccagcacagt ggcggccgct cgagtctaga 6420
gggcccgcgg ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgcg 6480
taccggttag caattgtttt ttcgatgagt ttggacaaac cacaactaga atgcagtgaa 6540
aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 6600
gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg 6660
tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtacttaa gagggggaga 6720
ccaaagggcg agacgttaag gcctcacgtg acatgtgagc aaaaggccag caaaaggcca 6780
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 6840
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 6900
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 6960
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 7020
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 7080
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 7140
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 7200
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 7260
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 7320
ccggcaaaca aaccacgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 7380
cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 7440
gaacgaaaac tcacgttaag ggattttggt catgccgtct cagaagaact cgtcaagaag 7500
gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg 7560
gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg 7620
atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc 7680
caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg 7740
catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc 7800
cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg 7860
tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc 7920
atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc 7980
cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc 8040
tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc 8100
attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag 8160
ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag 8220
cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcataatatt 8280
attgaagcat ttatcagggt tcgtctcgtc ccggtctcct cccatgcatg 8330
<210> 8
<211> 5123
<212> DNA
<213> 人工序列(Unknown)
<400> 8
tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60
ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120
aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180
gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240
gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300
agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360
ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420
cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480
gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660
cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg 720
tcgtttagtg aaccgtcaga tcactagtag ctttattgcg gtagtttatc acagttaaat 780
tgctaacgca gtcagtgctc gactgatcac aggtaagtat caaggttaca agacaggttt 840
aaggaggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg 900
cacctattgg tcttactgac atccactttg cctttctctc cacagggaaa aaacaattga 960
caagtttgta caaaaaagca ggctccgaat tcaccggtgc cgccaccatg gggatcctcc 1020
cccccaagaa aaagaggaag gtatctagag gccgcagccg ccttttggaa gattttcgaa 1080
acaaccggta ccccaattta caactgcggg agattgctgg acatataatg gaattttccc 1140
aagaccagca tgggtccaga ttcattcagc tgaaactgga gcgtgccaca ccagctgagc 1200
gccagcttgt cttcaatgaa atcctccagg ctgcctacca actcatggtg gatgtgtttg 1260
gtaattacgt cattcagaag ttctttgaat ttggcagtct tgaacagaag ctggctttgg 1320
cagaacggat tcgaggccac gtcctgtcat tggcactaca gatgtatggc agccgtgtta 1380
tcgagaaagc tcttgagttt attccttcag accagcagaa tgagatggtt cgggaactag 1440
atggccatgt cttgaagtgt gtgaaagatc agaatggcaa tcacgtggtt cagaaatgca 1500
ttgaatgtgt acagccccag tctttgcaat ttatcatcga cgcgtttaag ggacaggtat 1560
ttgccttatc cacacatcct tatggctgcc gagtgattca gagaatcctg gagcactgtc 1620
tccctgacca gacactccct attttagagg agcttcacca gcacacagag cagctggtac 1680
aggatcaata tggaaattat gtaatccaac atgtactgga gcacggtcgt cctgaggata 1740
aaagcaaaat tgtagcagaa atccgaggca atgtacttgt attgagtcag cacaaatttg 1800
caagcaatgt tgtggagaag tgtgttactc acgcctcacg tacggagcgc gctgtgctca 1860
tcgacgaggt gtgcaccatg aacgacggtc cccacagtgc cttatacacc atgatgaagg 1920
accagtatgc caactacgtg gtccagaaga tgattgacgt ggcggagcca ggccagcgga 1980
agatcgtcat gcataagatc cggccccaca tcgcaactct tcgtaagtac acctatggca 2040
agcacattct ggccaagctg gagaagtact acatgaagaa cggtgttgac ttaggggggc 2100
cggccggagg aggtggaagc ggaggaggag gaagcggagg aggaggtagc ggacctaaga 2160
aaaagaggaa ggtggcggcc gctggatccc cttcagggca gatcagcaac caggccctgg 2220
ctctggcccc tagctccgct ccagtgctgg cccagactat ggtgccctct agtgctatgg 2280
tgcctctggc ccagccacct gctccagccc ctgtgctgac cccaggacca ccccagtcac 2340
tgagcgctcc agtgcccaag tctacacagg ccggcgaggg gactctgagt gaagctctgc 2400
tgcacctgca gttcgacgct gatgaggacc tgggagctct gctggggaac agcaccgatc 2460
ccggagtgtt cacagatctg gcctccgtgg acaactctga gtttcagcag ctgctgaatc 2520
agggcgtgtc catgtctcat agtacagccg aaccaatgct gatggagtac cccgaagcca 2580
ttacccggct ggtgaccggc agccagcggc cccccgaccc cgctccaact cccctgggaa 2640
ccagcggcct gcctaatggg ctgtccggag atgaagactt ctcaagcatc gctgatatgg 2700
actttagtgc cctgctgtca cagatttcct ctagtgggca gggaggaggt ggaagcggct 2760
tcagcgtgga caccagtgcc ctgctggacc tgttcagccc ctcggtgacc gtgcccgaca 2820
tgagcctgcc tgaccttgac agcagcctgg ccagtatcca agagctcctg tctccccagg 2880
agccccccag gcctcccgag gcagagaaca gcagcccgga ttcagggaag cagctggtgc 2940
actacacagc gcagccgctg ttcctgctgg accccggctc cgtggacacc gggagcaacg 3000
acctgccggt gctgtttgag ctgggagagg gctcctactt ctccgaaggg gacggcttcg 3060
ccgaggaccc caccatctcc ctgctgacag gctcggagcc tcccaaagcc aaggacccca 3120
ctgtctccat cgattgatta attaagaatt cgacccagct ttcttgtaca aagtggttga 3180
tatccagcac agtggcggcc gctcgagtct agagggcccg cggttcgaag gtaagcctat 3240
ccctaaccct ctcctcggtc tcgattctac gcgtaccggt tagcaattgt tttttcgatg 3300
agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg 3360
atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt 3420
gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa 3480
acctctacaa atgtggtact taagaggggg agaccaaagg gcgagacgtt aaggcctcac 3540
gtgacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 3600
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 3660
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 3720
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 3780
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 3840
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 3900
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 3960
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 4020
ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 4080
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacg ctggtagcgg 4140
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 4200
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 4260
ggtcatgccg tctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc 4320
gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc 4380
agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc 4440
acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc 4500
gccatgggtc acgacgagat cctcgccgtc gggcatgctc gccttgagcc tggcgaacag 4560
ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc 4620
ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt 4680
agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc 4740
aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc 4800
ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag 4860
ccacgatagc cgcgctgcct cgtcttgcag ttcattcagg gcaccggaca ggtcggtctt 4920
gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc 4980
gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc 5040
tgcgtgcaat ccatcttgtt caatcataat attattgaag catttatcag ggttcgtctc 5100
gtcccggtct cctcccatgc atg 5123
<210> 9
<211> 23
<212> DNA
<213> 人工序列(Unknown)
<400> 9
gatcatttga acgaggcaac tca 23
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Unknown)
<400> 10
tggacccttc ggatcactca 20
<210> 11
<211> 21
<212> DNA
<213> 人工序列(Unknown)
<400> 11
catgtacgtt gctatccagg c 21
<210> 12
<211> 21
<212> DNA
<213> 人工序列(Unknown)
<400> 12
ctccttaatg tcacgcacga t 21
Claims (5)
1.一种制备特异性靶向目标基因SATB1的sgRNA，其特征在于，sgRNA的序列如SEQ IDNO.1所示。
2.根据权利要求1所述的sgRNA在转录激活靶向SATB1基因中的应用，其特征在于，通过以下步骤实现：
（1）sgRNA寡核苷酸的设计和选择：
（2）利用sgRNA，合成可以连入pAC-sgRNA-5×PBSa真核表达载体的双链sgRNA寡聚核苷酸；
（3）将sgRNA构建至pAC-sgRNA-5×PBSa载体，鉴定并扩增：
（4）转染Hela细胞并转录激活SATB1：
（a）按照jetPRIME DNA & siRNA Transfection Reagent的操作手册，用 800ng脂质体装载的pAC-SATB1 sgRNA-5×PBSa 质粒、400ng的pmax-dCas9质粒以及800ng的pAC1393 -PUFa-P65-HSF1质粒，分别转染至Hela细胞；其中pmax-dCas9质粒的序列如SEQ No .7所示，pAC1393 -PUFa-P65-HSF1质粒的序列如SEQ No.8所示；
（b）转染48小时后收集转然后的Hela细胞，提取总RNA和总蛋白，Real time Q-PCR及Western Blot确认SATB1被转录激活。
3. 根据权利要求2所述的应用，其特征在于，步骤（1）具体操作为：在SATB1基因上选择特征为5’- N(19)GG或5’-N(20)GG-3’或5’-N(21)GG-3’的序列；sgRNA在SATB1基因上的靶向位点位于转录起始位点后200bp内；按以上原则，设计得到sgRNA，序列为SEQ No.1；
4.根据权利要求2所述的应用，其特征在于，步骤（2）具体操作为：
（a）根据选择的sgRNA，去除3’端NGG三个碱基，在其5’端加上CACCG得到正向寡核苷酸；根据选择的sgRNA，去除3’端NGG三个碱基，获得对应DNA的互补链，并且在其5’加上AAAC,3’端加上C得到反向寡核苷酸，上述正向、反向寡核苷酸序列见SEQ NO.2和SEQ NO.3；
（b）分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的sgRNA寡聚核苷酸变性、退火，合成可以连入pAC-sgRNA-5×PBSa真核表达载体的双链。
5.根据权利要求2所述的应用，其特征在于，步骤（3）具体操作为：
（a）将退火的sgRNA寡聚核苷酸双链与序列如SEQ NO.4所示的pAC-sgRNA-5×PBSa载体连接，获得序列如SEQ NO.5所示的pAC-SATB1 sgRNA-5×PBSa质粒；
（b）pAC-SATB1 sgRNA-5×PBSa质粒转化感受态细菌并涂 Amp+平板；
（c）对pAC-SATB1 sgRNA-5×PBSa质粒进行鉴定：挑选阳性克隆并用序列如SEQ IDNO.6所示的通用引物 U6 测序的方法鉴定阳性克隆，37℃摇床摇阳性克隆菌过夜并用抽提pAC-SATB1 sgRNA-5×PBSa 质粒。
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| 通过CRISPR/Cas9系统敲除人源PDE10A基因;梁振伟等;《基础医学与临床》;20140430;第34卷(第4期);第439-443页 |
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