CN108559745A - 基于CRISPR-Cas9技术提高B16F10细胞转染效率的方法 - Google Patents
基于CRISPR-Cas9技术提高B16F10细胞转染效率的方法 Download PDFInfo
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Abstract
本发明公开了基于CRISPR‑Cas9技术提高B16F10细胞转染效率的方法。本发明首先获得特异性靶向SQSTM1基因第二外显子的sgRNA序列；其次构建SQSTM1基因的sgRNA到慢病毒载体系统，该载体表达Cas9蛋白；最后将含有该sgRNA的CRISPR/Cas9慢病毒载体转染B16F10细胞，药筛后获得SQSTM1的敲除细胞株。转染GFP质粒实验发现，所获得的SQSTM1的B16F10敲除细胞株具有转染效率明显高于未敲除细胞的优势。本发明具有操作步骤简单、sgRNA靶向性好，且对SQSTM1基因切割效率高；并能明显提高B16F10细胞的转染效率，为基础研究提供更好的细胞工具。
Description
技术领域
本发明属于基因工程领域，更具体地说涉及基于CRISPR-Cas9技术提高B16F10细胞转染效率的方法。
背景技术
转染是将外源核酸（包含：质粒，siRNA等）导入真核细胞中并发挥其功能的一种技术。目前转染被广泛应用于生物研究当中。然而许多生物学功能研究和涉及基因治疗的多种药物治疗受限于细胞的转染效率。
B16F10细胞是一种小鼠黑色素瘤细胞株，易于培养；目前被用于小鼠体外Cas9gRNA活性验证，但目前该细胞转染效率不高，常用lipo2000脂转效率在10%左右，大大限制了后续gRNA活性验证的检测。
细胞内吞作用是调节细胞摄取质粒DNA的关键过程。重要的是，内吞作用可以促进诱导自噬作为细胞对胞外DNAs进入细胞而产生的防御机制。最近的报告显示，一些转染试剂，如磷酸钙沉淀（CPPs）和脂质体（阳离子脂质），用于基因传递诱导自噬。研究显示耗尽或敲除SQSTM1可以大大提高小鼠胚胎成纤维细胞（MEF）和胚胎干（ES）细胞和人宫颈癌（Hela）细胞的基因传递效率。这些数据提示敲除SQSTM1可能有助于建立新的方法提高哺乳动物细胞基因传递效率。
为了更好的提高难转染的B16F10细胞基因传递效率，本研究采用CRISPR/Cas9系统，通过设计针对SQSTM1基因序列的sgRNA片段，构建慢病毒sgRNA表达载体，从而特异性的靶向敲除SQSTM1基因。
参考文献
Schatzlein, A.G. (2001). Non-viral vectors in cancer gene therapy:principles and progress. Anticancer Drugs 12, 275-304.
Zabner, J., Fasbender, A.J., Moninger, T., Poellinger, K.A. and Welsh,M.J. (1995).Cellular and molecular barriers to gene transfer by a cationiclipid. J Biol Chem 270, 18997-9007.
Chen, X., Khambu, B., Zhang, H., Gao, W., Li, M., Chen, X., Yoshimori, T.and Yin,X.M. (2014). Autophagy induced by calcium phosphate precipitatestargets damaged endosomes. J Biol Chem 289, 11162-74.
Wileman, T. (2013). Autophagy as a defence against intracellularpathogens. Essays Biochem 55, 153-63。
发明内容
本发明的首要目的在于提供一种靶向敲除SQSTM1基因的sgRNA慢病毒载体。
本发明的另一目的在于提供靶向敲除SQSTM1基因的小鼠黑色素瘤细胞B16F10细胞的应用，提高B16F10细胞转染效率。
本发明的目的通过下述技术方案实现：一种靶向敲除SQSTM1的sgRNA选自DNA序列如下的SQSTM1 sgRNA：
SQSTM1sgRNA的序列如下：
SQSTM1sgRNAoligo1：5’- ACCG AGCTGAAGCGGCGGATCTCG -3’；
SQSTM1sgRNAoligo2：5’- AAAC CGAGATCCGCCGCTTCAGCT -3’；
一种靶向敲除SQSTM1的慢病毒载体，含有上述靶向敲除SQSTM1基因的sgRNA的DNA序列。
所述的靶向敲除SQSTM1基因的慢病毒载体的构建，包括如下步骤：
(1)使用BsmBI酶切慢病毒载体pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9，得到酶切后的pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9慢病毒载体；
( 2 )将上述靶向敲除SQSTM1基因的sgRNA的DNA序列磷酸化后与酶切后的pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9慢病毒载体连接，得到靶向敲除SQSTM1基因的pLenti-U6-SQSTM1spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9慢病毒载体。
步骤( 2 )中所述的DNA序列是将寡核苷酸链1( oligo1 )和寡核苷酸链2(oligo2 ) 变性、退火之后形成可以连入U6 真核表达载体的双链。
所述的靶向敲除SQSTM1基因的慢病毒载体在制备敲除SQSTM1基因的细胞株中的应用。
一种敲除SQSTM1基因的细胞株中的应用，是将所述的靶向敲除SQSTM1基因的慢病毒质粒转染目的细胞得到的。
所述的敲除SQSTM1基因的细胞株，具体是通过如下步骤构建得到：
（1）将所述的靶向敲除SQSTM1基因的慢病毒载体质粒转染目的细胞；
（2）转染48小时后，用2ug/ml嘌呤霉素（puromycin）筛选一周后活下来的细胞即为靶向敲除SQSTM1的混合细胞株，进一步培养得到混合克隆；
（3）铺单克隆细胞，并进一步放大培养；收集单克隆细胞，以其基因组DNA 为模板扩增包含所述靶序列的基因片段，TA克隆测序确认SQSTM1基因已经被敲除并获得基因敲除的细胞。
所述的目的细胞株优选为小鼠黑色素瘤细胞株。
所述的小鼠黑色素瘤细胞株优选为小鼠黑色素瘤细胞株B16F10。
本发明相对于现有技术具有如下的优点及效果：
本发明提供SQSTM1基因的sgRNA能有效靶向敲除SQSTM1基因，将其构建入pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9慢病毒载体，该载体能靶向敲除SQSTM1基因，简单快速得到靶向敲除SQSTM1基因的细胞株，从而有利于提高难转染细胞株的转染效率。
附图说明
图1 为本发明实施例中使用的载体质粒pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9的质粒图谱；
图2为本发明实施例中使用的载体质粒pEGFP-N1的质粒图谱；
图3 为本发明实施例中B16F10细胞转染含有SQSTM1-sgRNA的活性鉴定测序图；
图4 为本发明实施例中鉴定到的切割活性较高的一个单克隆细胞株3#的序列与小鼠SQSTM1基因组序列比对结果图；
图5为本发明实施例中单克隆细胞株3#与对照空细胞株（B16F10）的转染效率对比图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
1 .靶向敲除SQSTM1慢病毒质粒构建
1 .1sgRNA寡核苷酸链合成
使用CRISPR在线设计工具( http://crispr .mit .edu/ )根据评分系统，在SQSTM1的二号外显子上设计1个20bp的sgRNA，并通过BLAST验证无非特异性基因。编码链模板5′端添加ACCG，非编码链模板3′端添加AAAC，与BsmBI酶切后形成的粘性末端互补，设计1对CRISPR寡核苷酸链，见表1, 表1 SQSTM1靶向位点及sgRNA寡核苷酸序列
1 .2载体构建
1.2.1使用BsmBI酶切2μg pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9质粒，2h，37℃，酶切体系
1 .2.2使用GENRY胶回收试剂盒纯化酶切质粒产物，按说明书进行操作
1 .2.3磷酸化并退火sgRNA, 酶连体系
PCR仪器退火程序：37℃ 30min，95℃维持5min，每分钟降低5℃至25℃，4℃维持。
1.2.4将退火形成的sgRNA双链和酶切后的pLenti-U6-spgRNA v2.0-CMV-Puro-
P2A-3Flag-spCas9载体直接连接，室温下，10min。
1.2.5将连接后的质粒转化至感受态细胞DH5α中，均匀涂至LB固体培养基平板中，置于37℃培养箱中培养12-16小时，单个菌落即可出现。
1 .3挑取单个菌落扩大培养并质粒小提。
1.4测序鉴定质粒构建成功，并命名为pLenti-U6-SQSTM1spgRNA v2.0-CMV-
Puro-P2A-3Flag-spCas9。
1.5 筛选稳定细胞株
用含10%胎牛血清的高糖DMEM培养基于5%CO2，37℃恒温培养B16F10细胞(购于美国ATCC细胞库)。取对数期细胞以2×10^5/孔接种到12孔板培养。待细胞融合度达到60%~70%时更换成Opti-MEM培养基，1 小时后将敲除质粒2μg 经Lipo2000试剂，转染到B16F10细胞中，转染48小时后，向每孔加入嘌呤霉素( 2μg/ml )，隔天换液，并保持培养基的嘌呤霉素浓度恒定，筛选阳性克隆细胞。所得细胞株命名为B16F10- SQSTM1。并使用有限稀释法挑选B16F10- SQSTM1单克隆敲除细胞株。
2 .稳定细胞株鉴定及基因打靶方式鉴定
以各组敲除SQSTM1的单克隆细胞株基因组DNA为模板，设计针对SQSTM1第二外显子的引物，序列如SEQ ID NO.4~ NO.7进行两轮巢式PCR扩增。用rSQSTM1-4-1N和rSQSTM1-4-2N进行PCR，得到799bp的片段a，以a为模板，用rSQSTM1-4-3N和rSQSTM1-4-4N进行PCR，得到387bp的片段b，该片段电泳后，胶回收用于测序，测序引物为hSQSTM1-4-3N，表2为测序引物序列。如测序结果打靶位点附近出现双峰的情况，则可能为打靶成功。如测序结果SQSTM1基因靶位点附近发生非三倍数的碱基插入或碱基缺失，导致阅读框移码突变，则可判断为SQSTM1基因敲除。鉴定出sgRNA是有活性的（图3），并且找到1株具有SQSTM1基因插入突变的单克隆细胞株(图4)。
表2 测序引物名称及序列。
实施例2
1 . SQSTM1敲除细胞株转染
1 .1SQSTM1敲除细胞株和对照空细胞株（B16F10）分别铺于6孔板1个孔；
1.2第二天细胞汇合度达80%的时候安排转染；转染时配制DNA和转染试剂：6孔板质粒时每孔比例为质粒pEGFP-N1（图2）：转染试剂（lipofectmine 2000）=5ug：5ul；将稀释好DNA及转染试剂，常温孵育5min；将稀释好的DNA和转染试剂混匀，常温孵育20min；
将孔板中的培养基弃上清，把残液吸干净，加入新鲜不含双抗的培养基；将混好的质粒及转染试剂滴加于孔中；转染6小时后，更换新鲜完全培养基；转染48小时后，显微镜下观察，3#克隆转染效率明显高于未进行敲除的空细胞（图5）。
序列表
<110> 和元生物技术(上海)股份有限公司
<120> 基于CRISP-Cas9技术提高B16F10细胞转染效率的方法
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cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt 2280
aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt 2340
cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta 2400
acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg 2460
agacgatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg 2520
atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg 2580
cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg 2640
aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag 2700
aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct 2760
ggggaatata acgtctcagt ttcagagcta tgctggaaac agcatagcaa gttgaaataa 2820
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ggatccatta 2880
gacgcgtggg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 2940
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 3000
actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 3060
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 3120
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 3180
ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 3240
cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 3300
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 3360
atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 3420
cagatcgcct gccatccacg ctgttttgac ctccatagaa gacaccgact ctactagagg 3480
atcgctagcg ctaccggact cagatctcga gctcaagctt cgaattcgcc accatgaccg 3540
agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc 3600
tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca 3660
tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca 3720
aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg 3780
aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc 3840
tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt 3900
ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg 3960
tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct 4020
ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg 4080
aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc ggctccggag 4140
ccacgaactt ctctctgtta aagcaagcag gcgacgtgga agaaaacccc ggtccggcta 4200
gcgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca 4260
aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc 4320
cagcagccga caagaagtac tccattgggc tcgatatcgg cacaaacagc gtcggctggg 4380
ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt caaagttctg ggcaataccg 4440
atcgccacag cataaagaag aacctcattg gcgccctcct gttcgactcc ggggagaccg 4500
ccgaagccac gcggctcaaa agaacagcac ggcgcagata tacccgcaga aagaatcgga 4560
tctgctacct gcaggagatc tttagtaatg agatggctaa ggtggatgac tctttcttcc 4620
ataggctgga ggagtccttt ttggtggagg aggataaaaa gcacgagcgc cacccaatct 4680
ttggcaatat cgtggacgag gtggcgtacc atgaaaagta cccaaccata tatcatctga 4740
ggaagaagct tgtagacagt actgataagg ctgacttgcg gttgatctat ctcgcgctgg 4800
cgcatatgat caaatttcgg ggacacttcc tcatcgaggg ggacctgaac ccagacaaca 4860
gcgatgtgga caaactcttt atccaactgg ttcagactta caatcagctt ttcgaagaga 4920
acccgatcaa cgcatccgga gttgacgcca aagcaatcct gagcgctagg ctgtccaaat 4980
cccggcggct cgaaaacctc atcgcacagc tccctgggga gaagaagaac ggcctgtttg 5040
gtaatcttat cgccctgtca ctcgggctga cccccaactt taaatctaac ttcgacctgg 5100
ccgaagatgc caagcttcaa ctgagcaaag acacctacga tgatgatctc gacaatctgc 5160
tggcccagat cggcgaccag tacgcagacc tttttttggc ggcaaagaac ctgtcagacg 5220
ccattctgct gagtgatatt ctgcgagtga acacggagat caccaaagct ccgctgagcg 5280
ctagtatgat caagcgctat gatgagcacc accaagactt gactttgctg aaggcccttg 5340
tcagacagca actgcctgag aagtacaagg aaattttctt cgatcagtct aaaaatggct 5400
acgccggata cattgacggc ggagcaagcc aggaggaatt ttacaaattt attaagccca 5460
tcttggaaaa aatggacggc accgaggagc tgctggtaaa gcttaacaga gaagatctgt 5520
tgcgcaaaca gcgcactttc gacaatggaa gcatccccca ccagattcac ctgggcgaac 5580
tgcacgctat cctcaggcgg caagaggatt tctacccctt tttgaaagat aacagggaaa 5640
agattgagaa aatcctcaca tttcggatac cctactatgt aggccccctc gcccggggaa 5700
attccagatt cgcgtggatg actcgcaaat cagaagagac catcactccc tggaacttcg 5760
aggaagtcgt ggataagggg gcctctgccc agtccttcat cgaaaggatg actaactttg 5820
ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc tctgctgtac gagtacttca 5880
cagtttataa cgagctcacc aaggtcaaat acgtcacaga agggatgaga aagccagcat 5940
tcctgtctgg agagcagaag aaagctatcg tggacctcct cttcaagacg aaccggaaag 6000
ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat tgaatgtttc gactctgttg 6060
aaatcagcgg agtggaggat cgcttcaacg catccctggg aacgtatcac gatctcctga 6120
aaatcattaa agacaaggac ttcctggaca atgaggagaa cgaggacatt cttgaggaca 6180
ttgtcctcac ccttacgttg tttgaagata gggagatgat tgaagaacgc ttgaaaactt 6240
acgctcatct cttcgacgac aaagtcatga aacagctcaa gaggcgccga tatacaggat 6300
gggggcggct gtcaagaaaa ctgatcaatg gcatccgaga caagcagagt ggaaagacaa 6360
tcctggattt tcttaagtcc gatggatttg ccaaccggaa cttcatgcag ttgatccatg 6420
atgactctct cacctttaag gaggacatcc agaaagcaca agtttctggc cagggggaca 6480
gtcttcacga gcacatcgct aatcttgcag gtagcccagc tatcaaaaag ggaatactgc 6540
agaccgttaa ggtcgtggat gaactcgtca aagtaatggg aaggcataag cccgagaata 6600
tcgttatcga gatggcccga gagaaccaaa ctacccagaa gggacagaag aacagtaggg 6660
aaaggatgaa gaggattgaa gagggtataa aagaactggg gtcccaaatc cttaaggaac 6720
acccagttga aaacacccag cttcagaatg agaagctcta cctgtactac ctgcagaacg 6780
gcagggacat gtacgtggat caggaactgg acatcaatcg gctctccgac tacgacgtgg 6840
atcatatcgt gccccagtct tttctcaaag atgattctat tgataataaa gtgttgacaa 6900
gatccgataa aaatagaggg aagagtgata acgtcccctc agaagaagtt gtcaagaaaa 6960
tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat cacacaacgg aagttcgata 7020
atctgactaa ggctgaacga ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa 7080
ggcagcttgt tgagacacgc cagatcacca agcacgtggc ccaaattctc gattcacgca 7140
tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga ggtgaaagtt attactctga 7200
agtctaagct ggtctcagat ttcagaaagg actttcagtt ttataaggtg agagagatca 7260
acaattacca ccatgcgcat gatgcctacc tgaatgcagt ggtaggcact gcacttatca 7320
aaaaatatcc caagcttgaa tctgaatttg tttacggaga ctataaagtg tacgatgtta 7380
ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc caccgctaag tacttctttt 7440
acagcaatat tatgaatttt ttcaagaccg agattacact ggccaatgga gagattcgga 7500
agcgaccact tatcgaaaca aacggagaaa caggagaaat cgtgtgggac aagggtaggg 7560
atttcgcgac agtccggaag gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg 7620
aagtacagac cggaggcttc tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc 7680
tgatcgcacg caaaaaagat tgggacccca agaaatacgg cggattcgat tctcctacag 7740
tcgcttacag tgtactggtt gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa 7800
gcgtcaagga actgctgggc atcacaatca tggagcgatc aagcttcgaa aaaaacccca 7860
tcgactttct ggaggcgaaa ggatataaag aggtcaaaaa agacctcatc attaagcttc 7920
ccaagtactc tctctttgag cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg 7980
agctgcagaa aggtaacgag ctggcactgc cctctaaata cgttaatttc ttgtatctgg 8040
ccagccacta tgaaaagctc aaagggtctc ccgaagataa tgagcagaag cagctgttcg 8100
tggaacaaca caaacactac cttgatgaga tcatcgagca aataagcgag ttctccaaaa 8160
gagtgatcct cgccgacgct aacctcgata aggtgctttc tgcttacaat aagcacaggg 8220
ataagcccat cagggagcag gcagaaaaca ttatccactt gtttactctg accaacttgg 8280
gcgcgcctgc agccttcaag tacttcgaca ccaccataga cagaaagcgg tacacctcta 8340
caaaggaggt cctggacgcc acactgattc atcagtcaat tacggggctc tatgaaacaa 8400
gaatcgacct ctctcagctc ggtggagaca agcgtcctgc tgctactaag aaagctggtc 8460
aagctaagaa aaagaaatga gtcgactcta gaccgcgtct ggaacaatca acctctggat 8520
tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 8580
ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 8640
tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 8700
caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 8760
accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 8820
ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 8880
tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc 8940
tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 9000
ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 9060
acgagtcgga tctccctttg ggccgcctcc ccgcctggaa ttaattctgc agtcgagacc 9120
tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct 9180
ggctagaagc acaagaggag gaggaggtgg gtttttccag tcacacctca ggtaccttta 9240
agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagagggga 9300
ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac 9360
acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca 9420
ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt agaagaggcc 9480
aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg 9540
gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga 9600
gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 9660
gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga 9720
tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg 9780
agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 9840
ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 9900
cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat 9960
tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggcctt gacattgcta 10020
gcgttttacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 10080
tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 10140
aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 10200
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 10260
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 10320
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 10380
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 10440
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 10500
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 10560
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 10620
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 10680
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 10740
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 10800
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 10860
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 10920
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 10980
aacaaaccac cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 11040
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 11100
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 11160
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 11220
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 11280
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 11340
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 11400
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 11460
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 11520
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 11580
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 11640
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 11700
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 11760
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 11820
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 11880
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 11940
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 12000
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 12060
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 12120
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 12180
ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcaacttg 12240
tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 12300
gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 12360
gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc caccccatac 12420
cctattacca ctgccaatta cctagtggtt tcatttactc taaacctgtg attcctctga 12480
attattttca ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagtttt 12540
taaagaaatt gtatttgtta aatatgtact acaaacttag tagt 12584
<210> 9
<211> 12284
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 9
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga actacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgatacgcgt gcggccgccc ccttcaccga gggcctattt 2220
cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt 2280
aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt 2340
cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta 2400
acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgcg 2460
ctacacaagt cgtagtctgt ttcagagcta tgctggaaac agcatagcaa gttgaaataa 2520
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ggatccatta 2580
gacgcgtggg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 2640
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 2700
actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 2760
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 2820
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 2880
ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 2940
cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 3000
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 3060
atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 3120
cagatcgcct gccatccacg ctgttttgac ctccatagaa gacaccgact ctactagagg 3180
atcgctagcg ctaccggact cagatctcga gctcaagctt cgaattcgcc accatgaccg 3240
agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc 3300
tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca 3360
tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca 3420
aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg 3480
aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc 3540
tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt 3600
ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg 3660
tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct 3720
ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg 3780
aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc ggctccggag 3840
ccacgaactt ctctctgtta aagcaagcag gcgacgtgga agaaaacccc ggtccggcta 3900
gcgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca 3960
aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc 4020
cagcagccga caagaagtac tccattgggc tcgatatcgg cacaaacagc gtcggctggg 4080
ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt caaagttctg ggcaataccg 4140
atcgccacag cataaagaag aacctcattg gcgccctcct gttcgactcc ggggagaccg 4200
ccgaagccac gcggctcaaa agaacagcac ggcgcagata tacccgcaga aagaatcgga 4260
tctgctacct gcaggagatc tttagtaatg agatggctaa ggtggatgac tctttcttcc 4320
ataggctgga ggagtccttt ttggtggagg aggataaaaa gcacgagcgc cacccaatct 4380
ttggcaatat cgtggacgag gtggcgtacc atgaaaagta cccaaccata tatcatctga 4440
ggaagaagct tgtagacagt actgataagg ctgacttgcg gttgatctat ctcgcgctgg 4500
cgcatatgat caaatttcgg ggacacttcc tcatcgaggg ggacctgaac ccagacaaca 4560
gcgatgtgga caaactcttt atccaactgg ttcagactta caatcagctt ttcgaagaga 4620
acccgatcaa cgcatccgga gttgacgcca aagcaatcct gagcgctagg ctgtccaaat 4680
cccggcggct cgaaaacctc atcgcacagc tccctgggga gaagaagaac ggcctgtttg 4740
gtaatcttat cgccctgtca ctcgggctga cccccaactt taaatctaac ttcgacctgg 4800
ccgaagatgc caagcttcaa ctgagcaaag acacctacga tgatgatctc gacaatctgc 4860
tggcccagat cggcgaccag tacgcagacc tttttttggc ggcaaagaac ctgtcagacg 4920
ccattctgct gagtgatatt ctgcgagtga acacggagat caccaaagct ccgctgagcg 4980
ctagtatgat caagcgctat gatgagcacc accaagactt gactttgctg aaggcccttg 5040
tcagacagca actgcctgag aagtacaagg aaattttctt cgatcagtct aaaaatggct 5100
acgccggata cattgacggc ggagcaagcc aggaggaatt ttacaaattt attaagccca 5160
tcttggaaaa aatggacggc accgaggagc tgctggtaaa gcttaacaga gaagatctgt 5220
tgcgcaaaca gcgcactttc gacaatggaa gcatccccca ccagattcac ctgggcgaac 5280
tgcacgctat cctcaggcgg caagaggatt tctacccctt tttgaaagat aacagggaaa 5340
agattgagaa aatcctcaca tttcggatac cctactatgt aggccccctc gcccggggaa 5400
attccagatt cgcgtggatg actcgcaaat cagaagagac catcactccc tggaacttcg 5460
aggaagtcgt ggataagggg gcctctgccc agtccttcat cgaaaggatg actaactttg 5520
ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc tctgctgtac gagtacttca 5580
cagtttataa cgagctcacc aaggtcaaat acgtcacaga agggatgaga aagccagcat 5640
tcctgtctgg agagcagaag aaagctatcg tggacctcct cttcaagacg aaccggaaag 5700
ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat tgaatgtttc gactctgttg 5760
aaatcagcgg agtggaggat cgcttcaacg catccctggg aacgtatcac gatctcctga 5820
aaatcattaa agacaaggac ttcctggaca atgaggagaa cgaggacatt cttgaggaca 5880
ttgtcctcac ccttacgttg tttgaagata gggagatgat tgaagaacgc ttgaaaactt 5940
acgctcatct cttcgacgac aaagtcatga aacagctcaa gaggcgccga tatacaggat 6000
gggggcggct gtcaagaaaa ctgatcaatg gcatccgaga caagcagagt ggaaagacaa 6060
tcctggattt tcttaagtcc gatggatttg ccaaccggaa cttcatgcag ttgatccatg 6120
atgactctct cacctttaag gaggacatcc agaaagcaca agtttctggc cagggggaca 6180
gtcttcacga gcacatcgct aatcttgcag gtagcccagc tatcaaaaag ggaatactgc 6240
agaccgttaa ggtcgtggat gaactcgtca aagtaatggg aaggcataag cccgagaata 6300
tcgttatcga gatggcccga gagaaccaaa ctacccagaa gggacagaag aacagtaggg 6360
aaaggatgaa gaggattgaa gagggtataa aagaactggg gtcccaaatc cttaaggaac 6420
acccagttga aaacacccag cttcagaatg agaagctcta cctgtactac ctgcagaacg 6480
gcagggacat gtacgtggat caggaactgg acatcaatcg gctctccgac tacgacgtgg 6540
atcatatcgt gccccagtct tttctcaaag atgattctat tgataataaa gtgttgacaa 6600
gatccgataa aaatagaggg aagagtgata acgtcccctc agaagaagtt gtcaagaaaa 6660
tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat cacacaacgg aagttcgata 6720
atctgactaa ggctgaacga ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa 6780
ggcagcttgt tgagacacgc cagatcacca agcacgtggc ccaaattctc gattcacgca 6840
tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga ggtgaaagtt attactctga 6900
agtctaagct ggtctcagat ttcagaaagg actttcagtt ttataaggtg agagagatca 6960
acaattacca ccatgcgcat gatgcctacc tgaatgcagt ggtaggcact gcacttatca 7020
aaaaatatcc caagcttgaa tctgaatttg tttacggaga ctataaagtg tacgatgtta 7080
ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc caccgctaag tacttctttt 7140
acagcaatat tatgaatttt ttcaagaccg agattacact ggccaatgga gagattcgga 7200
agcgaccact tatcgaaaca aacggagaaa caggagaaat cgtgtgggac aagggtaggg 7260
atttcgcgac agtccggaag gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg 7320
aagtacagac cggaggcttc tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc 7380
tgatcgcacg caaaaaagat tgggacccca agaaatacgg cggattcgat tctcctacag 7440
tcgcttacag tgtactggtt gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa 7500
gcgtcaagga actgctgggc atcacaatca tggagcgatc aagcttcgaa aaaaacccca 7560
tcgactttct ggaggcgaaa ggatataaag aggtcaaaaa agacctcatc attaagcttc 7620
ccaagtactc tctctttgag cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg 7680
agctgcagaa aggtaacgag ctggcactgc cctctaaata cgttaatttc ttgtatctgg 7740
ccagccacta tgaaaagctc aaagggtctc ccgaagataa tgagcagaag cagctgttcg 7800
tggaacaaca caaacactac cttgatgaga tcatcgagca aataagcgag ttctccaaaa 7860
gagtgatcct cgccgacgct aacctcgata aggtgctttc tgcttacaat aagcacaggg 7920
ataagcccat cagggagcag gcagaaaaca ttatccactt gtttactctg accaacttgg 7980
gcgcgcctgc agccttcaag tacttcgaca ccaccataga cagaaagcgg tacacctcta 8040
caaaggaggt cctggacgcc acactgattc atcagtcaat tacggggctc tatgaaacaa 8100
gaatcgacct ctctcagctc ggtggagaca agcgtcctgc tgctactaag aaagctggtc 8160
aagctaagaa aaagaaatga gtcgactcta gaccgcgtct ggaacaatca acctctggat 8220
tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 8280
ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 8340
tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 8400
caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 8460
accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 8520
ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 8580
tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc 8640
tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 8700
ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 8760
acgagtcgga tctccctttg ggccgcctcc ccgcctggaa ttaattctgc agtcgagacc 8820
tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct 8880
ggctagaagc acaagaggag gaggaggtgg gtttttccag tcacacctca ggtaccttta 8940
agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagagggga 9000
ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac 9060
acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca 9120
ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt agaagaggcc 9180
aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg 9240
gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga 9300
gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 9360
gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga 9420
tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg 9480
agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 9540
ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 9600
cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat 9660
tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggcctt gacattgcta 9720
gcgttttacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 9780
tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 9840
aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 9900
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 9960
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 10020
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 10080
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 10140
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 10200
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 10260
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 10320
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 10380
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 10440
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 10500
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 10560
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 10620
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 10680
aacaaaccac cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 10740
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 10800
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 10860
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 10920
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 10980
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 11040
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 11100
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 11160
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 11220
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 11280
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 11340
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 11400
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 11460
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 11520
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 11580
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 11640
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 11700
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 11760
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 11820
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 11880
ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcaacttg 11940
tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 12000
gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 12060
gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc caccccatac 12120
cctattacca ctgccaatta cctagtggtt tcatttactc taaacctgtg attcctctga 12180
attattttca ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagtttt 12240
taaagaaatt gtatttgtta aatatgtact acaaacttag tagt 12284
Claims (6)
1.基于CRISPR-Cas9提高B16F10细胞转染效率的方法，其特征在于，首先设计获得特异性靶向SQSTM1基因第二外显子的sgRNA；其次构建SQSTM1基因的sgRNA到慢病毒载体；然后将此慢病毒载体转染B16F10细胞，药筛后得到SQSTM1基因敲除细胞株。
2.根据权利要求1所述的特异性靶向SQSTM1基因第二外显子的sgRNA，其DNA 序列如SEQ ID NO.1所示，所述sgRNA 在SQSTM1基因上的靶序列是唯一的。
3.根据权利要求2所述的特异性靶向SQSTM1基因第二外显子的sgRNA，其引物包括：
针对SQSTM1基因的靶点位于第二外显子上的引物对：
SQSTM1 sgRNA oligo1：如SEQ ID NO.2所示；
SQSTM1 sgRNA oligo2：如SEQ ID NO.3所示。
4.根据权利要求1所述的构建SQSTM1基因的sgRNA到慢病毒载体，是一种靶向敲除SQSTM1基因的CRISPR-Cas9 重组表达慢病毒载体pLenti-U6-SQSTM1spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9，其特征在于：含有特异性靶向SQSTM1基因第二外显子的sgRNA 和Cas9 蛋白的重组表达载体。
5.根据权利要求4所述的重组表达载体的骨架载体的序列如序列表中SEQ ID NO.8所示；
根据权利要求1所述的CRISPR-Cas9 靶向敲除B16F10细胞SQSTM1基因及其特异性的sgRNA，其特征在于包括如下步骤：
(1) 提供sgRNA，所述sgRNA 在SQSTM1基因上的靶序列符合5’-N(19)G 的序列排列规则，所述sgRNA在SQSTM1基因上的靶序列位于基因的外显子，所述sgRNA在SQSTM1基因上的靶序列位于不同的各种剪切形式的共有外显子上，所述sgRNA在SQSTM1基因上的靶序列是唯一的，并且所述sgRNA在SQSTM1上的靶位点序列如序列表SEQ ID NO.1序列所示，在所述sgRNA在SQSTM1上的靶位点序列的5’-端加上ACCG序列合成得到正向寡核苷酸即Forwardoligo ；获得sgRNA 在SQSTM1上的靶位点序列的互补链，并且在互补链的5’-端加上AAAC序列得到反向寡核苷酸即Reverse oligo ；将合成的1 对互补的sgRNA 寡聚核苷酸的Forward oligo 和Reverse oligo 成对变性、退火，退火之后形成可以连入包含U6启动子的慢病毒表达载体的双链sgRNA寡聚核苷酸；
(2)线性化序列如序列表SEQ ID NO.8 所示的pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9质粒；将退火的双链sgRNA 寡聚核苷酸与线性化的载体pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9连接获得携带含相应靶序列的sgRNA 寡聚核苷酸的表达载体pLenti-U6-SQSTM1spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9质粒，转化感受态细菌并涂Amp+ 平板，挑取单克隆并用通用引物U6 通过测序鉴定出阳性克隆，并对所述阳性克隆摇菌、提取质粒；
(3)用所述携带有sgRNA 寡聚核苷酸和Cas9 基因的序列为SEQ ID NO.9的表达载体pLenti-U6-SQSTM1spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9质粒转染B16F10细胞；
(4)使用所述质粒转染小鼠B16F10细胞，转染48小时后用2ug/ml嘌呤霉素（puromycin）进行筛选后，进一步培养得到混合克隆；
(5)铺单克隆细胞，并进一步放大培养；收集单克隆细胞，以其基因组DNA 为模板扩增包含所述靶序列的基因片段，TA克隆测序确认SQSTM1基因已经被敲除并获得基因敲除的细胞。
6.根据权利要求1所述的敲除SQSTM1基因的细胞株，为B16F10细胞；其特征在于：是B16F10细胞中的SQSTM1基因缺失或插入核苷酸得到的细胞株，为如下的细胞株：sgRNA的切割后，导致SQSTM1基因的第二号外显子增加126个碱基；经过比对，蛋白翻译提前终止。
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