CN110760544A - 拆分型SpCas9慢病毒载体及其在干细胞基因编辑中的应用 - Google Patents

拆分型SpCas9慢病毒载体及其在干细胞基因编辑中的应用 Download PDF

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CN110760544A
CN110760544A CN201911098816.1A CN201911098816A CN110760544A CN 110760544 A CN110760544 A CN 110760544A CN 201911098816 A CN201911098816 A CN 201911098816A CN 110760544 A CN110760544 A CN 110760544A
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cas9
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孙志娟
刘德芳
罗天明
齐海龙
郭潇
年聚会
杨欢
孙忠杰
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Baoding Norway Technology Co Ltd
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Abstract

本发明涉及基因编辑技术领域，尤其涉及拆分型SpCas9慢病毒载体及其在干细胞基因编辑中的应用。本发明将SpCas9‑N‑Intein‑N和Intein‑C‑SpCas9‑C分别表达于两个慢病毒载体上，在侵染生物体后Intein‑N和Intein‑C互相识别形成完整的内含肽、进行自我剪切并把相邻的Cas9‑N和Cas9‑C连接成完整的Cas9，从而能够在体内实现对基因的编辑。实验表明，本发明的慢病毒载体可以有效感染人动员外周血来源的造血干细胞并对其进行基因编辑，编辑效率最高达50％。

Description

拆分型SpCas9慢病毒载体及其在干细胞基因编辑中的应用
技术领域
本发明涉及基因编辑技术领域，尤其涉及拆分型SpCas9慢病毒载体及其在干细胞基因编辑中的应用。
背景技术
CRISPR(clustered regularly interspaced short palindromic repeats)-Cas9 (CRIPSR associated protein 9)系统已经被广泛应用于各个科研领域，比如基因修饰、基因表达调控和表观遗传修饰等。CRISPR-Cas9系统最大的优点在于其应用的简便性：在特异性的单个引导RNA(single guide RNA,sgRNA)的引导下，Cas9几乎可以靶向人类基因组中的任何序列。但是，CRISPR-Cas9系统在人类疾病治疗方面的应用仍存在许多挑战，其中之一就是巨大的Cas9蛋白不便于病毒载体的运载。目前应用最广泛的Cas9蛋白是S.pyogenes Cas9 (SpCas9)。编码SpCas9蛋白的基因长约4kb(kilobase pair)，再加上sgRNA 及标志蛋白(比如荧光蛋白和抗药蛋白)的编码基因和驱动这些基因表达的启动子，总长超过5kb。这已经逼近慢病毒载体的运载上限，既严重降低了慢病毒的包装效率和病毒滴度，也严重影响了对目标细胞的感染效率。
基于自体造血干细胞移植的基因治疗已经开展了大规模的临床试验，针对的疾病包括重症免疫缺陷、地中海贫血、镰刀状贫血等各种遗传性血液系统疾病。目前，基因治疗的造血干细胞主要来源于病人的动员过的外周血，基因治疗的方式主要是利用慢病毒载体将突变基因的正确版本导入造血干细胞，从而达到治疗效果。CRISPR-Cas9可以高效切割基因组DNA，既可以对目标基因进行敲除，也可以对突变基因进行原位修正。因此，CRISPR-Cas9 在基因治疗方面有很大的应用前景。但是，如上所述，巨大的Cas9蛋白是 CRISPR-Cas9系统在基因治疗中应用的障碍。
对于SpCas9蛋白结构的研究，使得对其进行合理的改造成为可能。SpCas9 的蛋白结构呈双叶型，分别为α-螺旋叶和核酸酶叶。在与sgRNA结合之后，核酸酶叶相对于α-螺旋叶会发生很大程度的旋转，从而在这两叶结构之间形成一个带有正电荷的沟状结构以容纳带有负电荷的目标DNA。在与DNA结合的过程中，α-螺旋叶和核酸酶叶的相互作用是由目标DNA中介的，而不是由直接的蛋白-蛋白相互作用实现的。这一特性表明SpCas9的两叶结构可以被拆分为两个单独的同时又能重新组合成完整SpCas9的两段多肽。
蛋白质中的内含肽(intein)类似于mRNA中的内含子，在蛋白质被翻译出来后，内含肽可以将自己剪切掉并把相邻的两部分以肽键无缝连接从而产生最终的成熟的蛋白质。点状蓝藻(cyanobacteria Nostoc punctiforme)的DNA 聚合酶III的DnaE亚基由两个位于不同基因组位置的基因编码。其中，dnaE-n 按顺序编码DnaE和内含肽的氨基端片段(DnaE-N-Intein-N)，dnaE-c按顺序编码内含肽和DnaE的羧基端片段(Intein-C-DnaE-C)。Intein-N和Intein-C互相识别形成完整的内含肽、进行自我剪切并把相邻的DnaE-N和DnaE-C连接成完整的DnaE。因此，利用内含肽自我剪切的特性及SpCas9的结构特点，可以将SpCas9拆分为两部分，分别表达于不同的载体中。
发明内容
有鉴于此，本发明要解决的技术问题在于提供拆分型SpCas9慢病毒载体及其应用。该载体的感染效率较高。
本发明提供的拆分型SpCas9慢病毒载体，包括N端载体和C端载体：
所述N端载体包括顺序连接的如下元件：HIV 5’LTR、HIV-1ψ、RRE、 cPPT/CTS、Sv40NLS、FLAG、Cas9-N、DnaE Intein-N、P2A、EGFP、酶切位点、U6promoter、3’LTR；
所述C端载体包括顺序连接的如下元件：HIV 5’LTR、HIV-1ψ、RRE、 cPPT/CTS、DnaEIntein-C、Cas9-C、HA、Sv40 NLS、P2A、mCherry、3’LTR。
本发明利用SpCas9蛋白结构以及DnaE Intein的特性，将SpCas9在第573 号和574号氨基酸之间拆分为氨基端片段(SpCas9-N，1-573氨基酸)和羧基端片段(SpCas9-C，574-1368氨基酸)，分别与DnaE Intein的氨基端片段和羧基端片段形成融合蛋白，即SpCas9-N-Intein-N和Intein-C-SpCas9-C，并分别表达于两个慢病毒载体上，这两个载体被分别命名为N端载体和C端载体。
本发明所述的拆分型SpCas9慢病毒载体中，
所述Cas9-N的核苷酸序列如SEQ ID NO:1所示；
所述Cas9-C的核苷酸序列如SEQ ID NO:2所示；
所述DnaE Intein-N的核苷酸序列如SEQ ID NO:3所示；
所述DnaE Intein-C的核苷酸序列如SEQ ID NO:4所示。
本发明提供的病毒载体为一种基于HIV的改良型慢病毒载体，启动子位于5’LTR。该载体3’LTR的一部分被替换为MSCV(murine stem cell virus)3’ LTR的一部分，这一改进使得该载体比普通的基于HIV的慢病毒载体对 CD34+细胞的感染效率提高一倍。具体的，本发明所述的拆分型SpCas9慢病毒载体中，所述3’LTR包括HIV3’LTR、MSCV 3’LTR、HIV3’LTR三部分，其核苷酸序列如SEQ ID NO:5所示。所述HIV 5’LTR的核苷酸序列如SEQ ID NO:10所示。
在Cas9-N载体中，SpCas9-N-Intein-N的表达以绿色荧光蛋白(GFP)为标志，中间以能够自我剪切的猪捷申病毒2A多肽(P2A)连接，整体结构为 SpCas9-N-Intein-N-P2A-GFP。sgRNA由人核内小RNA U6的启动子驱动表达，并以相反的方向置于SpCas9-N-Intein-N-P2A-GFP之后。本发明所述的拆分型 SpCas9慢病毒载体中，所述U6 promoter核苷酸序列如SEQ ID NO:6所示。所述P2A的核苷酸序列如SEQ ID NO:7所示。所述GFP的核苷酸序列如SEQ ID NO:8所示。
在Cas9-C载体中，Intein-C-SpCas9-C的表达以红色荧光蛋白(mCherry) 为标志，中间以能够自我剪切的猪捷申病毒2A多肽(P2A)连接，整体结构为Intein-C-SpCas9-C-P2A-mCherry。所述mCherry的核苷酸序列如SEQ ID NO:9所示。
Sv40 NLS的核苷酸序列如SEQ ID NO:11所示。
HIV-1ψ-RRE的核苷酸序列如SEQ ID NO:12所示。
cPPT/CTS的核苷酸序列如SEQ ID NO:13所示。
FLAG的核苷酸序列如SEQ ID NO:14所示。
HA的核苷酸序列如SEQ ID NO:15所示。
N端载体上的酶切位点用于插入靶向sgRNA片段，本发明中，酶切位点采用BsmB I。
一些具体实施例中，N端载体的全长序列如SEQ ID NO:16所示，C端载体的全长序列如SEQ ID NO:17所示。
本发明还提供了包装有所述拆分型SpCas9慢病毒载体的慢病毒。
本发明所述的慢病毒，其包装载体为Delta8.9，外壳蛋白载体为VSVG。
所述慢病毒的包装方法为：将慢病毒载体、包装载体、外壳蛋白载体(质量比为4：3：1)转染HEK293T细胞系，培养后收集病毒。
所述转染DNA总质量与转染试剂的质量-体积比为1μg：3μL。
本发明所述的拆分型SpCas9慢病毒载体、或所述的慢病毒在细胞基因编辑中的应用。
本发明的慢病毒载体或慢病毒能够用于在侵染生物体后，Intein-N和 Intein-C互相识别形成完整的内含肽、进行自我剪切并把相邻的Cas9-N和 Cas9-C连接成完整的Cas9，因而能够在体内实现对基因的编辑。本发明中，所述细胞为动物细胞。而本发明提供的载体比普通的基于HIV的慢病毒载体对CD34+细胞的感染效率提高一倍。在一些具体实施例中，所述细胞为CD34+ 细胞；具体为造血干细胞。
本发明还提供了一种基因编辑方法，包括：
I)、将所述的拆分型SpCas9慢病毒载体中，N端载体的酶切位点处插入靶向sgRNA序列；
II)、包装所述的C端载体和I)所得的载体，获得慢病毒；
III)、以II)所得的慢病毒侵染宿主，获得基因编辑的细胞。
在本发明实施例中，进行编辑验证的基因为GYPA和AAVS1。
具体的，sgRNA序列的插入方法包括：
设计靶向基因的sgRNA及退火用的寡聚核苷酸链，并进行退火反应得到 sgRNA双链DNA；对N端载体进行酶切，获得线性化的N端载体；连接线性化N端载体和sgRNA双链DNA。
CRISPR/Cas9切割DNA后产生双链断裂。在没有模板DNA的情况下，双链断裂由非同源重组的末端连接机制修复，产生随机的插入或删除突变。在有模板DNA的情况下，双链断裂由同源臂介导的同源重组机制修复，将外源DNA插入到特定的基因组位置，实现精确的基因编辑。
本发明中的拆分型Cas9慢病毒载体可以在细胞中高效切割基因组DNA，产生双链断裂。其后，既可以不添加模板DNA以达到基因突变的目的，又可以添加模板DNA对基因进行精确的编辑。
为了实现对基因进行精准编辑，本发明所述的方法中还包括在步骤III中添加打靶载体。
所述打靶载体中包含两个同源臂和夹在其中的外源序列。
本发明所述基因编辑方法制得的宿主细胞。
对于经本发明所述基因编辑方法制得的宿主细胞，特别是经编辑的 CD34+细胞，能够用于疾病的治疗。针对的疾病包括重症免疫缺陷、地中海贫血、镰刀状贫血等各种遗传性血液系统疾病。
本发明还提供了一种制剂，其包括所述的宿主细胞。
本发明提供的制剂中经本发明所述基因编辑方法制得的宿主细胞悬浮于制剂的溶剂中。
本发明将SpCas9-N-Intein-N和Intein-C-SpCas9-C分别表达于两个慢病毒载体上，在侵染生物体后Intein-N和Intein-C互相识别形成完整的内含肽、进行自我剪切并把相邻的Cas9-N和Cas9-C连接成完整的Cas9，从而能够在体内实现对基因的编辑。实验表明，本发明的慢病毒载体可以有效感染人动员外周血来源的造血干细胞并对其进行基因编辑，编辑效率最高达50％。
附图说明
图1示本发明载体与原理示意图：其中A为拆分型SpCas9载体的工作原理示意图；B为拆分型SpCas9系统中两个载体片段，Cas9-N和Cas9-C的结构；
图2示编辑效果：其中A为拆分型SpCas9载体感染CD34+细胞的结果； B为通过T7E1方法检测的拆分型SpCas9载体的切割效率：GYPA检测片段全长659bp，经SpCas9切割后，产生442bp和217bp条带，AAVS1检测片段全长687bp，经SpCas9切割后，产生477bp和210bp条带；、C示部分经编辑的细胞的测序结果；其中WT示正常人基因组序列，其余数字为大肠杆菌克隆的编号；
图3示CRISPR/Cas9切割DNA后的修复机制。
具体实施方式
本发明提供了拆分型SpCas9慢病毒载体及其在干细胞基因编辑中的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试材皆为普通市售品，皆可于市场购得。
G-CSF动员的外周血单核细胞来源的CD34+细胞购买于Fred Hutchinson CancerResearch Center；
转染试剂Fugene6，生产厂商为Promega，货号为E2692；
DNA聚合酶，生产厂商为Tadara，货号为R010B；
T7 Endonuclease I，生产厂商为NEB，货号为M0302；
BsmBI，生产厂商为NEB，货号为R0580；
CD34+细胞培养基成分：StemSpanSFEM II，SR1 500nM，UM171 35nM，Dexamethasone 100nM，SCF 100ng/mL，FLT3L 100ng/mL，IL-3 20ng/mL，IL-6 20ng/mL。
StemSpanSFEM II，生产厂商为StemCell Technologies，货号为09655；
SR1，生产厂商为Thermo Fisher Scientific，货号为182706；
UM171，生产厂商为Thermo Fisher Scientific，货号为501365017；
Dexamethasone，生产厂商为Sigma Aldrich，货号为D2915；
SCF，生产厂商为Peprotech，货号为300-07；
FLT3L，生产厂商为Peprotech，货号为300-19；
IL-3，生产厂商为Peprotech，货号为200-03；
IL-6，生产厂商为Peprotech，货号为200-06。
下面结合实施例，进一步阐述本发明：
实施例
1、制备拆分型SpCas9载体；
(1)Cas9-C载体可以直接使用，不需要额外的分子克隆过程；
(2)设计靶向GYPA和AAVS1基因的sgRNA及退火用的寡聚核苷酸链(表1)，并进行退火反应得到sgRNA双链DNA；
表1 sgRNA、引物及退火寡核苷酸链
GYPA sgRNA靶向序列 atctttgtattactattgtc
GYPA sgRNA正向退火寡核苷酸链 caccgatctttgtattactattgtc
GYPA sgRNA反向退火寡核苷酸链 caccgagcattaagtaccactgagg
GYPA上游检测引物 cattagaaatgagaaggtccatggc
GYPA下游检测引物 aaactggatttggccataaatactg
AAVS1 sgRNA靶向序列 gcaaactctccaagtgacc
AAVS1 sgRNA正向退火寡核苷酸链 caccgagcaaactctccaagtgacc
AAVS1 sgRNA反向退火寡核苷酸链 aaacggtcacttggagagtttgct
AAVS1上游检测引物 cttcttctgctgacccctgtgctttc
AAVS1下游检测引物 gcttggctcggcctcccacctccatc
(3)对Cas9-N进行BsmBI酶切反应。酶切后线性化的Cas9-N具有与退火后sgRNA双链DNA互补的粘性末端；
(4)对(2)、(3)中的sgRNA双链DNA与线性化的Cas9-N进行连接反应，得到表达靶向GYPA和AAVS1的sgRNA的Cas9-N载体，命名为 Cas9-N-sgRNA。
2、制备Cas9-N-sgRNA和Cas9-C慢病毒；
(1)HEK293T按照1.6×105/cm2的密度接种到培养皿中；
(2)第二天进行转染。慢病毒载体：包装载体：外壳蛋白载体＝4μg：3μg： 1μg。DNA总质量：转染试剂体积＝1μg：3μL。8小时后换新鲜的培养基。
(3)转染后48小时收集病毒，用0.45μm滤器过滤后既可用于感染细胞。
3、感染CD34+细胞
(1)将Cas9-N-sgRNA和Cas9-C病毒溶液按1：1混合，并重悬CD34+ 细胞，细胞密度为4×105/mL。将细胞悬液转移到6孔板中进行离心感染，4mL/ 孔。离心感染条件：500g，2小时，30℃。
(2)将细胞离心收集后，重悬于培养基中培养3天。
4、流式细胞术检测感染效率
将细胞离心并重悬于5％FBS PBS中，用流式细胞仪检测GFP+mCherry+ 细胞的比例。
5、T7E1检测切割效率
(1)提取细胞基因组，以表1中的检测引物，通过PCR反应产生检测片段。对每个sgRNA，都以未感染的细胞基因组为对照组。
(2)将PCR片段进行变性、退火处理。
(3)用T7 Endonuclease1处理(2)中的PCR片段。
(4)凝胶电泳检测切割条带，并分析切割效率。
6、测序检测切割效率
(1)将5、(1)中的PCR片段连接到中间载体上，随机挑取大肠杆菌克隆进行测序。
(2)以人类基因组序列为参照，分析测序结果，计算突变效率，结果如表2：
表2突变效率
靶向基因 测序克隆总数(个) 突变克隆数量(个) 突变比例
GYPA 15 6 40％
AAVS1 12 6 50％
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 保定诺未科技有限公司
<120> 拆分型SpCas9慢病毒载体及其在干细胞基因编辑中的应用
<130> MP1919469
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1716
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 60
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680
aagcagctga aagaggacta cttcaagaaa atcgag 1716
<210> 2
<211> 2385
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca 60
taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag 120
gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag 180
gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg 240
cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag 300
cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc 360
atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg 420
tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt 480
aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg 540
cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga 600
cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc 660
cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg 720
tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg 780
tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac 840
aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa 900
gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc 960
cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag 1020
gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag 1080
atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg 1140
aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac 1200
aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg 1260
ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac 1320
aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc 1380
gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc 1440
aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg 1500
tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat 1560
atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag 1620
aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc 1680
ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag 1740
tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc 1800
ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac 1860
ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg 1920
ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg 1980
aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag 2040
cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc 2100
agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc 2160
tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt 2220
accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg 2280
aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc 2340
ggcctgtacg agacacggat cgacctgtct cagctgggag gcgac 2385
<210> 3
<211> 306
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgcctgagct acgagaccga gatcctgacc gtggagtacg gcctgctgcc tatcggcaag 60
atcgtggaga agagaatcga gtgcaccgtg tacagcgtgg acaacaacgg caacatctac 120
acccagcctg tggcccagtg gcacgacaga ggcgagcagg aggtgttcga gtactgcctg 180
gaggacggca gcctgatcag agccaccaag gaccacaagt tcatgaccgt ggacggccag 240
atgctgccta tcgacgagat cttcgagaga gagctggacc tgatgagagt ggacaacctg 300
cctaac 306
<210> 4
<211> 105
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgatcaaga tcgccaccag aaagtacctg ggcaagcaga acgtgtacga catcggcgtg 60
gagagagacc acaacttcgc cctgaagaac ggcttcatcg ccagc 105
<210> 5
<211> 612
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg attgaaagac 60
cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata 120
cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg 180
gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg 240
tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg 300
ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg 360
cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg 420
gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtagtggcga gccctcagat 480
gctgcatata agcagctgct ttttgcttgt actggggtct ctctggttag accagatctg 540
agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 600
ttgagtgctt ca 612
<210> 6
<211> 265
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cggtgtttcg tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta 60
cggtaagcat atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa 120
attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa 180
ttctaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa 240
ataggccctc ttcctgcccg acctt 265
<210> 7
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggatccggcg caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct 60
ggaccg 66
<210> 8
<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tctggcgagg gcgagggcga cgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 9
<211> 711
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60
gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120
cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180
ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240
cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300
gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360
ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420
atgcagaaga aaaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480
gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540
gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600
aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660
cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta a 711
<210> 10
<211> 634
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
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 agca 634
<210> 11
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cctaagaaga agagaaaggt g 21
<210> 12
<211> 1420
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag 60
gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc 120
aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa 180
gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa 240
atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc 300
tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct 360
tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt 420
gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca 480
aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag 540
atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat 600
taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg 660
gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt 720
caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca 780
atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca 840
agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg 900
ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt 960
ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag 1020
aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag 1080
aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta 1140
acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag 1200
gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac 1260
cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag 1320
aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac 1380
ggtatcgatg gatctacaaa tggcagtatt catccacaat 1420
<210> 13
<211> 117
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata 60
gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaatttt 117
<210> 14
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gactacaagg acgacgacga caag 24
<210> 15
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tacccttacg acgtgcctga ctacgcc 27
<210> 16
<211> 6336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
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
aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa 1200
ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag 1260
agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc 1320
ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga 1380
caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa 1440
cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct 1500
gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc 1560
atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt 1620
tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata 1680
cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa 1740
ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata 1800
aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt 1860
tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca 1920
accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga 1980
gacagatcca ttcgattagt gaacggatct cgacggtatc gatggatcta caaatggcag 2040
tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa 2100
tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa 2160
ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttgggga attcgccacc 2220
atgggcccta agaagaagag aaaggtggcc gccgccgact acaaggacga cgacgacaag 2280
ggcatccacg gcgtgcctgc cgccgacaag aagtacagca tcggcctgga catcggcacc 2340
aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag 2400
gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc 2460
gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc 2520
agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg 2580
gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac 2640
gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc 2700
accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg 2760
atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac 2820
ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac 2880
cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct 2940
gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag 3000
aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag 3060
agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac 3120
gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc 3180
aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc 3240
aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc 3300
ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac 3360
cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac 3420
aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg 3480
aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag 3540
atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg 3600
aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc 3660
cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc 3720
accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag 3780
cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg 3840
ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga 3900
atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc 3960
aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag 4020
tgcctgagct acgagaccga gatcctgacc gtggagtacg gcctgctgcc tatcggcaag 4080
atcgtggaga agagaatcga gtgcaccgtg tacagcgtgg acaacaacgg caacatctac 4140
acccagcctg tggcccagtg gcacgacaga ggcgagcagg aggtgttcga gtactgcctg 4200
gaggacggca gcctgatcag agccaccaag gaccacaagt tcatgaccgt ggacggccag 4260
atgctgccta tcgacgagat cttcgagaga gagctggacc tgatgagagt ggacaacctg 4320
cctaacggat ccggcgcaac aaacttctct ctgctgaaac aagccggaga tgtcgaagag 4380
aatcctggac cgcaattgat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 4440
atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc tggcgagggc 4500
gagggcgacg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 4560
cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 4620
taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 4680
caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 4740
ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 4800
ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 4860
gccgacaagc agaagaacgg catcaaggcg aacttcaaga tccgccacaa catcgaggac 4920
ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 4980
ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 5040
aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 5100
gacgagctgt acaagtaact cgagaaaaaa aagcaccgac tcggtgccac tttttcaagt 5160
tgataacgga ctagccttat ttaaacttgc tatgctgttt ccagcatagc tcttaaacag 5220
agacgttcca ggttcgtctc cggtgtttcg tcctttccac aagatatata aagccaagaa 5280
atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa 5340
aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt 5400
tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa 5460
atatgaagga atcatgggaa ataggccctc ttcctgcccg accttttaat taagaattat 5520
caagcttatc gataccgtcg agacctggaa aaacatggag caatcacaag tagcaataca 5580
gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt 5640
ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc 5700
cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat 5760
atccttgatc tgtggattga aagaccccac ctgtaggttt ggcaagctag cttaagtaac 5820
gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta 5880
ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc 5940
ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag cagtttctag 6000
agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg 6060
aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat 6120
aaaagagccc acaacccctc actcggcgcg ccagtcctcc gatagactgc gtcgcccggg 6180
tacccgtagt ggcgagccct cagatgctgc atataagcag ctgctttttg cttgtactgg 6240
gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 6300
gcttaagcct caataaagct tgccttgagt gcttca 6336
<210> 17
<211> 6438
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
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
aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa 1200
ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag 1260
agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc 1320
ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga 1380
caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa 1440
cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct 1500
gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc 1560
atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt 1620
tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata 1680
cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa 1740
ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata 1800
aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt 1860
tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca 1920
accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga 1980
gacagatcca ttcgattagt gaacggatct cgacggtatc gatggatcta caaatggcag 2040
tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa 2100
tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa 2160
ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttgggga attcgccacc 2220
atgatcaaga tcgccaccag aaagtacctg ggcaagcaga acgtgtacga catcggcgtg 2280
gagagagacc acaacttcgc cctgaagaac ggcttcatcg ccagctgctt cgactccgtg 2340
gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg 2400
aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat 2460
atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc 2520
tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc 2580
tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca 2640
atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac 2700
gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat 2760
agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg 2820
cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac 2880
atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc 2940
gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa 3000
caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat 3060
gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg 3120
gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc 3180
agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag 3240
atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac 3300
aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag 3360
agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg 3420
atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg 3480
aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc 3540
aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc 3600
aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg 3660
cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc 3720
tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg 3780
aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg 3840
gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc 3900
gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag 3960
ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc 4020
gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag 4080
agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc 4140
atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg 4200
cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc 4260
gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg 4320
gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt 4380
gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag 4440
agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg 4500
gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg 4560
ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc 4620
accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca 4680
cggatcgacc tgtctcagct gggaggcgac gcctaccctt acgacgtgcc tgactacgcc 4740
agcctgggca gcggcagccc taagaagaag agaaaggtgg aggaccctaa gaagaagaga 4800
aaggtggacg gatccggcgc aacaaacttc tctctgctga aacaagccgg agatgtcgaa 4860
gagaatcctg gaccgcaatt gatggtgagc aagggcgagg aggataacat ggccatcatc 4920
aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag 4980
atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg 5040
accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc 5100
tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc 5160
gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc 5220
caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac 5280
ttcccctccg acggccccgt aatgcagaag aaaaccatgg gctgggaggc ctcctccgag 5340
cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag 5400
gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag 5460
ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac 5520
accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag 5580
ctgtacaagt aactcgagtg ctaccgttaa ttaagaatta tcaagcttat cgataccgtc 5640
gagacctgga aaaacatgga gcaatcacaa gtagcaatac agcagctacc aatgctgatt 5700
gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca cctcaggtac 5760
ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg 5820
ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat ctgtggattg 5880
aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg 5940
aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag agacagcaga 6000
atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac 6060
agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc 6120
agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc 6180
ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct 6240
cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtag tggcgagccc 6300
tcagatgctg catataagca gctgcttttt gcttgtactg gggtctctct ggttagacca 6360
gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 6420
cttgccttga gtgcttca 6438

Claims (10)

1.拆分型SpCas9慢病毒载体，包括N端载体和C端载体：
所述N端载体包括顺序连接的如下元件：HIV 5’LTR、HIV-1ψ、RRE、cPPT/CTS、Sv40 NLS、FLAG、Cas9-N、DnaEIntein-N、P2A、EGFP、酶切位点、U6promoter、3’LTR；
所述C端载体包括顺序连接的如下元件：HIV 5’LTR、HIV-1ψ、RRE、cPPT/CTS、DnaEIntein-C、Cas9-C、HA、Sv40 NLS、P2A、mCherry、3’LTR。
2.根据权利要求1所述的拆分型SpCas9慢病毒载体，其特征在于，
所述Cas9-N的核苷酸序列如SEQ ID NO:1所示；
所述Cas9-C的核苷酸序列如SEQ ID NO:2所示；
所述DnaEIntein-N的核苷酸序列如SEQ ID NO:3所示；
所述DnaEIntein-C的核苷酸序列如SEQ ID NO:4所示。
3.根据权利要求1或2所述的拆分型SpCas9慢病毒载体，其特征在于，
所述3’LTR包括HIV3’LTR、MSCV 3’LTR、HIV3’LTR三部分，其核苷酸序列如SEQ ID NO:5所示。
4.根据权利要求1～3任一项所述的拆分型SpCas9慢病毒载体，其特征在于，所述U6promoter核苷酸序列如SEQ ID NO:6所示。
5.包装有权利要求1～4任一项所述拆分型SpCas9慢病毒载体的慢病毒。
6.权利要求1～4任一项所述的拆分型SpCas9慢病毒载体、权利要求5所述的慢病毒在细胞基因编辑中的应用。
7.一种基因编辑方法，其特征在于，包括：
I)、将权利要求1～4任一项所述的拆分型SpCas9慢病毒载体中，N端载体的酶切位点处插入靶向sgRNA序列；
II)、包装权利要求1～4任一项所述的C端载体和I)所得的载体，获得慢病毒；
III)、以II)所得的慢病毒侵染宿主，获得基因编辑的细胞。
8.根据权利要求7所述的基因编辑方法，其特征在于，还包括在步骤III中添加打靶载体。
9.权利要求7或8所述基因编辑方法制得的宿主细胞。
10.一种制剂，其包括权利要求9所述的宿主细胞。
CN201911098816.1A 2019-11-12 2019-11-12 拆分型SpCas9慢病毒载体及其在干细胞基因编辑中的应用 Pending CN110760544A (zh)

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