CN107142282A - 一种利用CRISPR/Cas9在哺乳动物细胞中实现大片段DNA定点整合的方法 - Google Patents

一种利用CRISPR/Cas9在哺乳动物细胞中实现大片段DNA定点整合的方法 Download PDF

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CN107142282A
CN107142282A CN201710220878.XA CN201710220878A CN107142282A CN 107142282 A CN107142282 A CN 107142282A CN 201710220878 A CN201710220878 A CN 201710220878A CN 107142282 A CN107142282 A CN 107142282A
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ccr5
cas9
crispr
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黄翔
何祖勇
刘小凤
刘小红
陈瑶生
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National Sun Yat Sen University
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Abstract

本发明公开了一种利用CRISPR/Cas9在哺乳动物细胞中实现大片段DNA定点整合的方法，包括如下步骤：(1)靶向CCR5基因的CRISPR/Cas9打靶系统的构建：打靶系统由Cas9/sgRNA表达载体和打靶供体组成，所述打靶供体包括启动子、荧光标记基因、中间含有gRNA靶序列的与目的基因的同源序列的设计；(2)CRISPR/Cas9打靶系统共转染哺乳动物细胞，转染后分选稳定表达荧光的细胞，进行单细胞克隆培养；(3)单细胞克隆PCR鉴定检测定点整合效率，获得高效定点定向整合的哺乳动物细胞。本发明能实现1个6kb以上大片段DNA在哺乳动物细胞中的定点定向整合。

Description

一种利用CRISPR/Cas9在哺乳动物细胞中实现大片段DNA定点 整合的方法
技术领域
本发明涉及基因工程技术领域，具体地，涉及一种利用CRISPR/Cas9在哺乳动物细胞中实现大片段DNA定点整合的方法。
背景技术
哺乳动物细胞基因组中进行大片段外源基因的定点整合，通常采用的是双交换同源重组的方法，该技术的实施步骤为通过构建包含目的基因两侧的两个同源臂的打靶供体，利用两个同源臂与目的基因进行双交换同源重组，从而将目的基因替换成两个同源臂之间的外源DNA片段来实现的。但是该技术存在如下不足：双交换同源重组的效率往往较低，一般为10-6；采用该技术实现定点整合的外源DNA片段相对较小，一般为2kb以下。具体原因可能是因为两个同源臂的物理距离越大越不利于在目的基因两侧同时进行同源重组交换的发生导致。
发明内容
本发明的目的是为了克服现有技术的上述不足，提供一种在哺乳动物细胞中实现大片段DNA定点整合的方法。本发明能实现约6kb大片段在哺乳动物细胞中的定点整合。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
一种利用CRISPR/Cas9在哺乳动物细胞中实现大片段DNA定点定向整合的方法，包括如下步骤：
(1)靶向CCR5基因的CRISPR/Cas9打靶系统的构建：打靶系统由Cas9/sgRNA表达载体和打靶供体组成，所述打靶供体包括启动子、荧光标记基因、中间含有gRNA靶序列的与目的基因的同源序列的设计；
(2)CRISPR/Cas9打靶系统共转染哺乳动物细胞，转染后分选稳定表达荧光的细胞，进行单细胞克隆培养；
(3)单细胞克隆PCR鉴定检测定点整合效率，获得高效定点定向整合的哺乳动物细胞。
优选地，所述靶向CCR5基因的CRISPR/Cas9靶向位点序列如SEQ ID NO：1所示。更优选地，所述靶向CCR5基因的gRNA正、负链序列分别如SEQ ID NO：2所示和SEQ ID NO：3所示。
作为优选实施方式，一种利用CRISPR/Cas9在哺乳动物细胞中实现大片段DNA定点定向整合的方法，包括如下步骤：
(1)构建靶向CCR5基因的打靶系统，打靶系统包括SEQ ID NO：4所示的pX330-CCR5质粒和SEQ ID NO：6所示的pEGFP-CCR5-HR打靶供体；
(2)将pX330-CCR5质粒、pEGFP-CCR5-HR打靶供体共转染HEK293T细胞，转染后利用流式分选稳定表达EGFP荧光的细胞，进行单细胞克隆培养；
(3)单细胞克隆进行PCR鉴定检测定点整合效率，获得定点定向整合的HEK293T细胞。
其中，pX330-CCR5质粒即为Cas9/sgRNA表达载体。
优选地，所述转染时，按照每孔HEK293T细胞150ng pX330-CCR5质粒、100ngCCR5-EGFP-Donor质粒(pEGFP-CCR5-HR)、1μL Lipofectamine 3000、1μLP3000TM和50μLOpti-MEM培养基配方添加进行转染。
优选地，当HEK293T细胞的汇合度至90％左右时，转染效率高。
与现有技术相比，本发明具有如下有益效果：
本发明首次采用单交换同源重组的原理将大片段外源DNA定点整合进哺乳动物细胞的基因组中，相较于传统的双交换同源重组策略，单交换可以较为高效的将大片段载体定点插入基因组。
本发明通过构建靶向CCR5基因的打靶供体，通过CRISPR/Cas9介导的一个打靶供体的定点定向整合，实现了1个6kb以上大片段DNA在哺乳动物细胞中的定点定向整合。
附图说明
图1为pX330载体的结构示意图。
图2为pEGFP-N1质粒结构示意图。
图3为pEGFP-CCR5-HR质粒结构示意图。
图4为gRNA位点设计以及打靶效率检测；(A)gRNA在CCR5基因上的位置示意图；(B)野生型和突变型的DNA序列以及各突变型的发生频率。
图5为单交换介导单种打靶供体定点整合至HEK293T细胞CCR5基因；(A)通过单交换或者非同源末端连接介导的单个打靶供体正向定点插入示意图；(B)通过非同源末端连接介导的单个打靶供体反向定点插入示意图；(C)PCR检测CRISPR/Cas9介导的单个打靶供体的定点插入方式均为正向插入，而非反向插入；(D)野生型和突变型的DNA序列以及各突变型的发生频率。
图6为CRISPR/Cas9介导单交换整合效率鉴定；(A)单细胞克隆荧光图；(B)单细胞克隆PCR检测CRISPR/Cas9介导单个打靶供体整合效率。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1
1、CCR5基因靶位点选择：根据CCR5基因序列以及gRNA基本设计原则，并借助软件分析(http://crispr.mit.edu/)，设计靶向人CCR5基因的gRNA，具体序列见表1。
表1.靶向CCR5基因的gRNA序列信息
名称 序列(5’to 3’)
CRISPR/Cas9靶向位点 CATCCTGATAAACTGCAAA
2、HEK293T细胞培养与转染：HEK293T细胞培养使用10％胎牛血清的DMEM高糖培养基，细胞以约1×105个/mL的密度接种于24孔细胞培养板中，待用。待细胞汇合度至90％左右时可用于转染，转染时按照每孔细胞150ng pX330-CCR5质粒、100ngCCR5-EGFP-Donor质粒(pEGFP-CCR5-HR)、1μL Lipofectamine 3000、1μLP3000TM和50μLOpti-MEM培养基配方添加转染，同时设置单转CCR5-EGFP-Donor质粒处理组和空白对照组。
其中，所述pX330-CCR5质粒、CCR5-EGFP-Donor质粒的构建方法如下。
pX330-CCR5质粒的构建步骤：合成CCR5-gRNA的正负序列，见下表2。
表2为CCR5-gRNA的正负序列
Primer名称 序列(5'to3')
CCR5-sgRNA F caccgCATCCTGATAAACTGCAAA
CCR5-sgRNA R aaacTTTGCAGTTTATCAGGATGc
通过Bbs1酶切位点克隆进pX330载体(见图1)，获得pX330-CCR5质粒(序列如SEQID NO：4所示)。
CCR5-EGFP-Donor质粒(pEGFP-CCR5-HR)的构建：合成长度为1640bp如SEQ ID NO：5所示的DNA片段，通过AseI酶切位点克隆进pEGFP-N1载体(见图2)，获得pEGFP-CCR5-HR质粒。pEGFP-CCR5-HR质粒的序列SEQ ID NO：6所示，结构图见图3。
3、细胞分选：转染后3天，将部分贴壁的HEK293T细胞用胰酶消化，1600rpm离心5min，去除上清，添加PBS重悬细胞，经50μm尼龙膜过滤到流式管中，流式细胞分选仪分选出荧光阳性细胞，接收至1.5ml离心管中，以备后续打靶效率鉴定。转染后20天，将荧光阳性细胞分选至1.5ml离心管和96孔培养皿，分别进行后续定点整合鉴定以及单细胞克隆培养整合效率检测。
4、测序分析：PCR扩增包含gRNA识别位点的CCR5基因片段，将目的片段克隆至T载，转化后挑选若干个单克隆菌落，进行测序分析。
5、结果：
(1)CRISPR/Cas9打靶效率鉴定：本技术设计了一条针对人CCR5基因的gRNA(图4.A)，并且通过T-A克隆确定该CRISPR/Cas9系统的打靶效率，结果表明挑选的10个单克隆菌落中，5个CCR5-sgRNA位点发生突变，打靶效率约为50％左右(图4.B)。
(2)检测单个打靶供体通过单交换机制实现定点整合的情况：我们首先设计了与CCR5基因组有同源序列且带有荧光标记基因的打靶供体，该同源序列分为左同源臂(LHA)和右同源臂(RHA)，在两段同源臂衔接处设计了gRNA的打靶序列位点，即CRISPR/Cas9可在基因组该位置及所构载体上同时产生双链断裂缺口(DSB)。根据单交换原理，打靶供体可被完整插入基因组序列中，打靶供体的插入分为正向插入(图5.A)和反向插入(图5.B)，正向插入才能确保gRNA同靶序列完整。具体操作为：将CRISPR/Cas9与带有EGFP标记基因的打靶供体转染HEK293T细胞，通过观察转染后不同时间段的荧光变化，结合流式分析数据，我们发现在转染后20天左右，荧光率维持在一个比较稳定的状态，表明该阶段绿色荧光细胞为打靶供体稳定整合细胞。流式分选稳定荧光表达的细胞后，提取基因组进行PCR鉴定，验证该打靶供体是否定点稳定整合进目的基因。我们分别在发生定点整合后的序列5端和3端设计了一对PCR引物，5端PCR扩增引物上游位于CCR5基因组上，下游位于所构打靶供体上，跨越左右同源臂，3端PCR扩增引物上游位于所构打靶供体上，下游位于CCR5基因组上，跨越左右同源臂，结果表明同时转染CRISPR/Cas9与打靶供体的基因组能够扩增出目的片段，表明发生了单交换，也就是打靶供体的定点整合，同时整合插入的类型为正向插入而非反向插入，而空白对照组与只转染打靶供体的基因组均不能扩增出该目的条带(图5.C)。进一步T-A克隆测序分析，结果表明CRISPR/Cas9介导单交换定点整合后，其打靶位点多存在一定程度的破坏，可能会阻碍进一步的多个打靶供体的整合(图5.D)。
(3)针对CRISPR/Cas9介导的单个打靶供体的整合，我们进一步通过单细胞克隆培养确定其整合效率。将流式分选后的EGFP阳性细胞分选至96孔细胞培养皿进行单细胞克隆培养，培养至7天左右，可观察到明显的带荧光的单细胞克隆细胞群(图6.A)。培养至20天左右，可提取相应单细胞克隆基因组进行PCR鉴定，PCR扩增结果表明，CRISPR/Cas9介导单个打靶供体的整合效率为10％左右(图6.B)。
SEQUENCE LISTING
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ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccc atcctgataa actgcaaagt tttagagcta gaaatagcaa gttaaaataa 300
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc 360
tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca attctgcaga 420
caaatggctc tagaggtacc cgttacataa cttacggtaa atggcccgcc tggctgaccg 480
cccaacgacc cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt 540
caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 600
ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag 660
tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 720
accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca 780
cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg 840
gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg 900
agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg 960
cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgacg 1020
ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 1080
gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 1140
gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta 1200
cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat 1260
aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacgataag 1320
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag 1380
tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag 1440
tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 1500
aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg 1560
aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag 1620
atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc 1680
ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac 1740
gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac 1800
agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc 1860
cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 1920
ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc 1980
ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat 2040
ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg 2100
agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg 2160
cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 2220
cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac 2280
atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga 2340
tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct 2400
gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac 2460
ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac 2520
ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc 2580
ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg 2640
cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg 2700
accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg 2760
atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag 2820
ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac 2880
gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg 2940
accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag 3000
aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg 3060
aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa 3120
gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag 3180
gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca 3240
ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac 3300
gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg 3360
aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag 3420
tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt 3480
aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt 3540
gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg 3600
gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc 3660
agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc 3720
gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc 3780
cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg 3840
gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag 3900
agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg 3960
ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg 4020
cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag 4080
agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc 4140
cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac 4200
gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc 4260
gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 4320
cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg 4380
gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag 4440
agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 4500
tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 4560
acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 4620
aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 4680
ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag 4740
gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg 4800
gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg 4860
gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 4920
aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc 4980
gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac 5040
gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 5100
ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 5160
tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 5220
gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 5280
caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc 5340
aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac 5400
gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 5460
ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag 5520
taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 5580
ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 5640
cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 5700
gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg 5760
agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc 5820
tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 5880
tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc 5940
atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg 6000
cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 6060
cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 6120
ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 6180
cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac 6240
ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 6300
tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat 6360
ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 6420
aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata 6480
gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 6540
cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 6600
ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata 6660
ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 6720
gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 6780
acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 6840
tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 6900
agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 6960
cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 7020
aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 7080
gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 7140
agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 7200
aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 7260
gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 7320
ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 7380
aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 7440
aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 7500
tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc 7560
agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 7620
ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 7680
ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 7740
ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 7800
acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 7860
agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 7920
ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 7980
cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 8040
gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 8100
cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 8160
gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 8220
caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 8280
aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 8340
tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 8400
gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 8460
ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt 8507
<210> 5
<211> 1640
<212> DNA
<213> 长度为1640bp的DNA片段
<400> 5
attaatccaa gctttgagtg gggtggctca tgcctgtaat cccagcattc taggaggctg 60
aggcaggagg atcgcttgag cccaggagtt cgagaccagc ctgggcaaca tagtgtgatc 120
ttgtatctat aaaaataaac aaaattagct tggtgtggtg gcgcctgtag tccccagcca 180
cttggagggg tgaggtgaga ggattgcttg agcccgggat ggtccaggct gcagtgagcc 240
atgatcgtgc cactgcactc cagcctgggc gacagagtga gaccctgtct cacaacaaca 300
acaacaacaa caaaaaggct gagctgcacc atgcttgacc cagtttctta aaattgttgt 360
caaagcttca ttcactccat ggtgctatag agcacaagat tttatttggt gagatggtgc 420
tttcatgaat tcccccaaca gagccaagct ctccatctag tggacaggga agctagcagc 480
aaaccttccc ttcactacaa aacttcattg cttggccaaa aagagagtta attcaatgta 540
gacatctatg taggcaatta aaaacctatt gatgtataaa acagtttgca ttcatggagg 600
gcaactaaat acattctagg actttataaa agatcacttt ttatttatgc acagggtgga 660
acaagatgga ttatcaagtg tcaagtccaa tctatgacat caattattat acatcggagc 720
cctgccaaaa aatcaatgtg aagcaaatcg cagcccgcct cctgcctccg ctctactcac 780
tggtgttcat ctttggtttt gtgggcaaca tgctggtcat cctcatcctg ataaactgca 840
aaaggctgaa gagcatgact gacatctacc tgctcaacct ggccatctct gacctgtttt 900
tccttcttac tgtccccttc tgggctcact atgctgccgc ccagtgggac tttgcaaata 960
caatgtgtca actcttgaca gggctctatt ttataggctt cttctctgga atcttcttca 1020
tcatcctcct gacaatcgat aggtacctgg ctgtcgtcca tgctgtgttt gctttaaaag 1080
ccaggacggt cacctttggg gtggtgacaa gtgtgatcac ttgggtggtg gctgtgtttg 1140
cgtctctccc aggaatcatc tttaccagat ctcaaaaaga aggtcttcat tacacctgca 1200
gctctcattt tccatacagt cagtatcaat tctggaagaa tttccagaca ttaaagatag 1260
tcatcttggg gctggtcctg ccgctgcttg tcatggtcat ctgctactcg ggaatcctaa 1320
aaactctgct tcggtgtcga aatgagaaga agaggcacag ggctgtgagg cttatcttca 1380
ccatcatgat tgtttatttt ctcttctggg ctccctacaa cattgtcctt ctcctgaaca 1440
ccttccagga attctttgac ctgaataatt gcagtagctc taacaggttg gaccaagcta 1500
tgcaggtgac agagactctt gggatgacgc actgctgcat caaccccatc atctatgcct 1560
ttgtcgggga gaagttcaga aactacctct tagtcttctt ccaaaagcac attgccaaac 1620
caagcaatga gtggattaat 1640
<210> 6
<211> 6367
<212> DNA
<213> pEGFP-CCR5-HR质粒
<400> 6
tagttattaa tccaagcttt gagtggggtg gctcatgcct gtaatcccag cattctagga 60
ggctgaggca ggaggatcgc ttgagcccag gagttcgaga ccagcctggg caacatagtg 120
tgatcttgta tctataaaaa taaacaaaat tagcttggtg tggtggcgcc tgtagtcccc 180
agccacttgg aggggtgagg tgagaggatt gcttgagccc gggatggtcc aggctgcagt 240
gagccatgat cgtgccactg cactccagcc tgggcgacag agtgagaccc tgtctcacaa 300
caacaacaac aacaacaaaa aggctgagct gcaccatgct tgacccagtt tcttaaaatt 360
gttgtcaaag cttcattcac tccatggtgc tatagagcac aagattttat ttggtgagat 420
ggtgctttca tgaattcccc caacagagcc aagctctcca tctagtggac agggaagcta 480
gcagcaaacc ttcccttcac tacaaaactt cattgcttgg ccaaaaagag agttaattca 540
atgtagacat ctatgtaggc aattaaaaac ctattgatgt ataaaacagt ttgcattcat 600
ggagggcaac taaatacatt ctaggacttt ataaaagatc actttttatt tatgcacagg 660
gtggaacaag atggattatc aagtgtcaag tccaatctat gacatcaatt attatacatc 720
ggagccctgc caaaaaatca atgtgaagca aatcgcagcc cgcctcctgc ctccgctcta 780
ctcactggtg ttcatctttg gttttgtggg caacatgctg gtcatcctca tcctgataaa 840
ctgcaaaagg ctgaagagca tgactgacat ctacctgctc aacctggcca tctctgacct 900
gtttttcctt cttactgtcc ccttctgggc tcactatgct gccgcccagt gggactttgc 960
aaatacaatg tgtcaactct tgacagggct ctattttata ggcttcttct ctggaatctt 1020
cttcatcatc ctcctgacaa tcgataggta cctggctgtc gtccatgctg tgtttgcttt 1080
aaaagccagg acggtcacct ttggggtggt gacaagtgtg atcacttggg tggtggctgt 1140
gtttgcgtct ctcccaggaa tcatctttac cagatctcaa aaagaaggtc ttcattacac 1200
ctgcagctct cattttccat acagtcagta tcaattctgg aagaatttcc agacattaaa 1260
gatagtcatc ttggggctgg tcctgccgct gcttgtcatg gtcatctgct actcgggaat 1320
cctaaaaact ctgcttcggt gtcgaaatga gaagaagagg cacagggctg tgaggcttat 1380
cttcaccatc atgattgttt attttctctt ctgggctccc tacaacattg tccttctcct 1440
gaacaccttc caggaattct ttgacctgaa taattgcagt agctctaaca ggttggacca 1500
agctatgcag gtgacagaga ctcttgggat gacgcactgc tgcatcaacc ccatcatcta 1560
tgcctttgtc ggggagaagt tcagaaacta cctcttagtc ttcttccaaa agcacattgc 1620
caaaccaagc aatgagtgga ttaatagtaa tcaattacgg ggtcattagt tcatagccca 1680
tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 1740
gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 1800
ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 1860
gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 1920
cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 1980
gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 2040
tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 2100
caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 2160
ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag 2220
atccgctagc gctaccggac tcagatctcg agctcaagct tcgaattctg cagtcgacgg 2280
taccgcgggc ccgggatcca ccggtcgcca ccatggtgag caagggcgag gagctgttca 2340
ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 2400
tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 2460
ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc 2520
agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 2580
ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 2640
gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 2700
acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 2760
acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 2820
acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 2880
gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca 2940
aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 3000
tcactctcgg catggacgag ctgtacaagt aaagcggccg cgactctaga tcataatcag 3060
ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa 3120
cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg 3180
ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 3240
tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata 3300
ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 3360
aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 3420
cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 3480
ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 3540
cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 3600
ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 3660
gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 3720
cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 3780
gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 3840
tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt 3900
gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 3960
gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 4020
tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 4080
cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 4140
tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg 4200
cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc 4260
gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat 4320
tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt 4380
cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac 4440
tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg 4500
tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc 4560
aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa 4620
tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc 4680
gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg 4740
aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg 4800
acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa 4860
atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg 4920
acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct 4980
tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc 5040
ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa 5100
cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat 5160
cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt 5220
cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg 5280
cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa 5340
acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg 5400
ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc 5460
agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat 5520
actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 5580
tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 5640
cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 5700
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 5760
tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 5820
gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 5880
gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 5940
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 6000
acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 6060
agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 6120
cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 6180
tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 6240
gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 6300
ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 6360
catgcat 6367

Claims (4)

1.一种利用CRISPR/Cas9在哺乳动物细胞中实现大片段DNA定点定向整合的方法，其特征在于，包括如下步骤：
(1)靶向CCR5基因的CRISPR/Cas9打靶系统的构建：打靶系统由Cas9/sgRNA表达载体和打靶供体组成，所述打靶供体包括启动子、荧光标记基因、中间含有gRNA靶序列的与目的基因的同源序列的设计；
(2) CRISPR/Cas9打靶系统共转染哺乳动物细胞，转染后分选稳定表达荧光的细胞，进行单细胞克隆培养；
(3)单细胞克隆PCR鉴定检测定点整合效率，获得高效定点定向整合的哺乳动物细胞。
2.根据权利要求1所述的方法，其特征在于，所述靶向CCR5基因的CRISPR/Cas9靶向位点序列如SEQ ID NO：1所示。
3.根据权利要求1所述的方法，其特征在于，所述靶向CCR5基因的gRNA正、负链序列分别如SEQ ID NO：2所示和SEQ ID NO：3所示。
4.根据权利要求3所述的方法，其特征在于，包括如下步骤：
(1) 构建靶向CCR5基因的打靶系统，打靶系统包括SEQ ID NO：4所示的pX330-CCR5质粒和SEQ ID NO：6所示的pEGFP-CCR5-HR打靶供体；
(2) 将pX330-CCR5质粒、pEGFP-CCR5-HR打靶供体共转染HEK293T细胞，转染后利用流式分选稳定表达EGFP荧光的细胞，进行单细胞克隆培养；
(3) 单细胞克隆进行PCR鉴定检测定点整合效率，获得定点定向整合的HEK293T细胞。
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