CN103224947A - 一种基因打靶系统 - Google Patents
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Abstract
一种基因打靶系统，由位点特异性切割核酸酶表达载体和打靶载体两部分组成，打靶载体包含2～10个供体DNA片段，每个供体DNA片段的5＇端和3＇端分别插入位点特异性切割核酸酶的识别序列，供体DNA由上游同源臂、下游同源臂和位于两者之间的外源DNA序列组成。位点特异性切割核酸酶表达载体是携带锌指核酸酶的表达载体、转录激活子样效应因子核酸酶的表达载体、RNA介导的核酸酶RNA:Cas9的表达载体中的任意一种。
Description
技术领域
本发明属于生物技术领域，具体涉及一种基因打靶系统。
技术背景
基因组的靶向修饰包括对基因组内源性基因序列的改造或者在基因组的特定位置插入外源性基因片段。这项技术为研究特定基因功能提供了有力工具，此外研究人员可以利用该技术建立特定的动物模型来进行基因功能研究或新药物的研发。传统的基因靶向修饰技术是依赖于自然状态下的同源重组（Homologous recombination，HR），效率非常低，大约为10-6，因而大大限制了该技术的应用。近几年来锌指核酸酶（Zinc finger nucleases,ZFN）、转录激活子样效应因子核酸酶（Transcription activator-like effector nucleases，TALEN）、RNA介导的Cas9核酸酶（RNA:Cas9）等位点特异性切割核酸酶技术的出现给基因组靶向修饰带来了希望。
ZFN、TALEN、RNA:Cas9等位点特异性切割核酸酶技术是近年来发展起来的一项新的技术，通过人工设计的ZFN、TALEN或sgRNA:Cas9在基因组DNA的特定位置切割产生双链断裂（DSB），继而通过细胞内源性的修复机制对断裂部位的基因进行修饰。同自然状态下的同源重组技术相比较，该技术可以使基因组靶向修饰的效率提高了103～105倍。位点特异性切割核酸酶介导的基因定点修饰已经在多种体外培养的细胞中获得成功，包括人的胚胎干细胞(embryonic stemcell,ES)和诱导性多能干细胞(induced pluripotent stem cells,iPS)、植物、果蝇、爪蟾、线虫、斑马鱼、小鼠、大鼠等的细胞，显示出该技术的广泛适用性，这将有力地推动基因靶向修饰技术的应用研究。
位点特异性切割核酸酶介导的基因靶向修饰需要将位点特异性切割核酸酶和供体DNA同时导入到靶细胞中，其中对供体DNA的需求量要高于位点特异性切割核酸酶，多数情况下将位点特异性切割核酸酶和供体DNA导入靶细胞的比例为1:5～1:10，如何有效提高将供体DNA导入到靶细胞中的效率成为影响基因靶向修饰效率的重要因素，尤其是对于那些转染效率低的细胞或者在体内基因靶向修饰中。
发明内容
本发明所要解决的技术问题在于克服上述供体DNA导入靶细胞效率的不足，提供一种能产生多个供体DNA片段、效率高的基因打靶系统。
解决上述技术问题所采用的技术方案是：由位点特异性切割核酸酶表达载体和打靶载体两部分组成。本发明的打靶载体包含2～10个供体DNA片段，每个供体DNA片段的5＇端和3＇端分别插入位点特异性切割核酸酶的识别序列，供体DNA由上游同源臂、下游同源臂和位于两者之间的外源DNA序列组成。上述的位点特异性切割核酸酶表达载体是携带锌指核酸酶的表达载体、转录激活子样效应因子核酸酶的表达载体、RNA介导的核酸酶RNA:Cas9的表达载体中的任意一种。
本发明的位点特异性切割核酸酶的识别序列是基因组上位点特异性切割核酸酶结合的长度为20bp～50bp的DNA序列。
本发明的外源DNA序列是长度为1bp～3000bp的DNA片段。
本发明的上游同源臂、下游同源臂是分别与位点特异性切割核酸酶识别位点上游和下游的部分基因组序列同源的两段长度为50bp～3000bp的DNA片段。
本发明的部分基因组序列是距离锌指核酸酶识别位点1bp～3000bp的DNA序列。
本发明采用由位点特异性切割核酸酶表达载体和携带多个供体DNA片段的打靶载体组成的基因打靶系统，在供体DNA片段之间引入了位点特异性切割核酸酶的识别序列，通过位点特异性切割核酸酶在细胞内对打靶载体的切割产生多个供体DNA片段，有效地提高供体DNA进入靶细胞的水平，从而提高基因打靶的效率。
附图说明
图1.是AAVS1ZFN表达载体结构图。
图2是AAVS11000F供体DNA结构图。
图3.是携带多个供体DNA片段的打靶载体结构图。
图4是pE1/EGFP/4×AAVS1-TSF-Donor靶向修饰效率检测。
图5是CCR5ZFN表达载体结构图。
图6是携带SNCA ZFN表达元件的腺病毒结构图。
图7是携带靶向SNCA位点的供体DNA的腺病毒结构图。
具体实施方式
下面结合附图和实施例对本发明进一步详细说明，但本发明不限于这些实施例。
实施例1
以靶向AAVS1位点的基因打靶系统为例，其组成如下：
1、表达AAVS1ZFN的锌指核酸酶表达载体
在质粒载体pshuttle(购买自安捷伦公司)的多克隆位点依次引入了真核启动子CMV、靶向AAVS1位点的锌指核酸酶AAVS1ZFN、转录终止信号TKpA，其中AAVS1ZFN包含左侧锌指核酸酶AAVS1ZFNL和右侧锌指核酸酶AAVS1ZFNR两部分，左侧锌指核酸酶AAVS1ZFNL与右侧锌指核酸酶AAVS1ZFNR之间由自剪切多肽T2A相连（即AAVS1ZFNL-T2A-AAVS1ZFNR），表达AAVS1ZFN的锌指核酸酶表达载体的结构见图1。本实施例的靶向AAVS1位点的锌指核酸酶AAVS1ZFN也可以用靶向AAVS1位点的转录激活子样效应因子核酸酶（AAVS1TALEN）、RNA介导的Cas9核酸酶（AAVS1gRNA-Cas9）中的任意一个替换。
AAVS1ZFNL-T2A-AAVS1ZFNR的序列见序列表<210>1到ttttga2166。
2、靶向AAVS1位点的打靶载体
在pshuttle的多克隆位点携带有串联重复的4个供体DNA片段AAVS1-1000F，每个供体DNA片段的5＇端和3＇端均插入了AAVS1ZFN的识别序列AAVS1-TSF，供体DNA片段由上游同源臂AAVS1UP、下游同源臂AAVS1DOWN和位于两者之间的外源DNA序列（ClaⅠ酶切位点，长度为6bp）组成，供体DNA片段结构见图2，上游同源臂是基因组上距离AAVS1-TSF1bp长度为500bp的DNA序列，下游同源臂是基因组上距离AAVS1TSF3bp长度为500bp的DNA序列，该靶向AAVS1位点的打靶载体命名为4×AAVS1-TSF-1000F，打靶载体结构见图3。
AAVS1-TSF的序列如下：
accccacagtggggccactagggacaggat
AAVS1-1000F的序列见序列表<210>3到cgtcctggca gggctg 1006。
4×AAVS1-TSF-1000F的序列见序列表<210>4到agatct 4206。
上述靶向AAVS1位点的基因打靶系统的构建方法步骤如下：
1、构建表达AAVS1ZFN的锌指核酸酶表达载体
在上海生工合成引物，序列如下：
P1:KBL for CGGATCCATCGATACTAGTGCGGCCGCGTCGACA;
P2:KBL reverse
GATCTGTCGACGCGGCCGCACTAGTATCGATGGATCCGGTAC;
将KBL for和KBL reverse在室温退火获得KBL片段(KpnⅠ-BamHⅠ-ClaⅠ-SpeⅠ-NotⅠ-SalⅠ-BglⅡ)。将KBL片段与经过KpnⅠ和BglⅡ酶切处理的pshuttle载体用SolutionⅠ试剂盒（Takara公司购买）连接，将连接产物转化到大肠杆菌DH5α细胞，并涂布于卡那抗性的培养基上，经过质粒提取、酶切鉴定获得阳性克隆，命名为pE1shuttle。
在上海生工合成CMV启动子和转录终止信号TKpA，
CMV序列见序列表<210>7到tactagt547。
TKpA序列见序列表<210>8到gcggccgc428。
用BamHⅠ和SpeⅠ酶切CMV片段，用SpeⅠ和NotⅠ酶切TKpA片段，通过琼脂糖凝胶电泳回收CMV和TKpA片段，与经过BamHⅠ和NotⅠ酶切处理的pE1shuttle载体用SolutionⅠ试剂盒（Takara公司购买）连接，连接条件如下：100ng/μl pE1shuttle载体0.5μl，100ng/μl CMV片段2μl，100ng/μl TkpA片段2μl，SolutionⅠ5μl，用水补至10μl，16℃连接过夜。经过质粒提取，酶切鉴定获得阳性克隆命名为pE1/CMV-TKpA。
在上海生工合成引物，序列如下：
P3:EHL for AATTgATCGATACTAGTt;
P4:EHL reverse AGCTaACTAGTATCGATc;
将EHL for和EHL reverse在室温退火获得EHL片段(EcoRⅠM-ClaⅠ-SpeⅠ-HindⅢM)。将EHL片段与经过EcoRⅠ和HindⅢ酶切处理的pUC19载体用SolutionⅠ试剂盒（Takara公司购买）连接，连接产物经过转化、质粒提取、酶切鉴定获得阳性克隆，命名为pUC19/EHL。
在上海生工合成锌指核酸酶基础结构，即NLS-Flag-FokⅠDD-T2A-NLS-HA-FokⅠRR，并在序列的两端引入ClaⅠ和SpeⅠ位点，在Flag和FokⅠDD之间引入HindⅢ和XhoⅠ位点用于插入左侧的锌指蛋白AAVS1ZFL，在HA和FokⅠRR之间引入KpnⅠ和BamHⅠ位点用于插入右侧的锌指蛋白AAVS1ZFR。锌指核酸酶基础结构的序列见序列表<210>11到aacttttgaa ctagt1525。
将合成的锌指核酸酶基础结构用ClaⅠ和SpeⅠ酶切处理后与经过同样酶切处理的pUC19/EHL用SolutionⅠ试剂盒（Takara公司购买）连接，将连接产物转化感受态的DH5α细胞，并涂布于含有100μg/ml的氨苄青霉素的LB平板中。挑取菌落接种到含有100μg/ml的氨苄青霉素的LB培养液中，14～16小时后，经碱性裂解法提取质粒DNA，通过酶切鉴定阳性克隆。将所获得阳性克隆命名为pUC19/EHL/FokⅠDD-T2A-FokⅠRR。
根据已经报道的靶向AAVS1位点的锌指蛋白的序列，在上海生工合成靶向AAVS1位点的一对锌指蛋白基因，分别命名为AAVS1ZFL和AAVS1ZFR。并且在AAVS1ZFL和AAVS1ZFR的两端分别引入了HindⅢ-SalⅠ和KpnⅠ-BglⅡ酶切位点。序列如下：
AAVS1ZFL的序列见序列表<210>12到attcatgtcg aca 353。
AAVS1ZFR的序列见序列表<210>13到attcatagat cta 353。
将AAVS1ZFL用HindⅢ和SalⅠ酶切，与经过HindⅢ和XhoⅠ酶切处理的pUC19/EHL/FokⅠDD-T2A-FokⅠRR载体用SolutionⅠ试剂盒（Takara公司购买）连接，经过转化、提取质粒、酶切鉴定，获得阳性克隆命名为pUC19/EHL/AAVS1ZFL-FokⅠDD-T2A-FokⅠRR。将AAVS1ZFR用KpnⅠ和BglⅡ酶切处理，与经过KpnⅠ和BamHⅠ酶切处理的pUC19/EHL/AAVS1ZFL-FokⅠDD-T2A-FokⅠRR载体用SolutionⅠ试剂盒（Takara公司购买）连接，经过转化、提取质粒、酶切鉴定，获得阳性克隆命名为pUC19/EHL/AAVS1ZFN（即pUC19/EHL/AAVS1ZFL-FokⅠDD-T2A-AAVS1ZFR-FokⅠRR）。靶向AAVS1的一对锌指核酸酶基因AAVS1ZFNL（AAVS1ZFL-FokⅠDD）和AAVS1ZFNR（AAVS1ZFR-FokⅠRR）之间通过自剪切多肽T2A相连。
将pUC19/EHL/AAVS1ZFN用ClaⅠ和SpeⅠ酶切并通过琼脂糖凝胶电泳回收AAVS1ZFN片段，与经过同样酶切处理的pE1/CMV-TKpA载体用SolutionⅠ试剂盒（Takara公司购买）连接，经过转化、质粒提取及酶切鉴定获得阳性克隆命名为pE1/AAVS1ZFN（如图1）。至此获得表达AAVS1ZFN的锌指核酸酶表达载体。
2、构建携带多个供体DNA片段的打靶载体
依据AAVS1位点的基因组序列设计引物用于扩增上游同源臂和下游同源臂，引物序列如下：
P5:AAVS1UP SalⅠfor AGTCGACcttcactcgctgggttcc
P6:AAVS1UP ClaⅠreverse AATCGATggaggggacagataaaag
P7:AAVS1DOWN ClaⅠfor AATCGATgtgacagaaaagccccatc
P8:AAVS1DOWN BglⅡreverse AAGATCTTTCTCGAGcagccctgccaggacggg
P9:AAVS1TSF for
TCGACcaccccacagtggggccactagggacaggattCTCGAGGGATCCA
P10:AAVS1TSF reverse
GATCTGGATCCCTCGAGaatcctgtccctagtggccccactgtggggtgG
P11:EGFP ClaⅠfor AATCGATATGGTGAGCAAGGGCGAGG
P12:EGFP SpeⅠreverse AACTAGTTTACTTGTACAGCTCGTCCAT
以pEGFP-N1（Clontech公司购买）为模板，P11/P12为引物，通过聚合酶链式反应扩增EGFP基因，聚合酶链式反应的条件按照
HS DNA聚合酶试剂盒的说明书进行。将EGFP基因克隆入pGEM-T Easy载体，经过酶切鉴定及测序鉴定获得阳性克隆命名为pGEMT/EGFP。将pGEMT/EGFP用ClaⅠ和SpeⅠ酶切，与经过同样酶切处理的pE1/CMV-TKpA用SolutionⅠ试剂盒（Takara公司购买）连接获得pE1/EGFP。
取P9（20μM）和P10（20μM）各15μl，于室温退火获得AAVS1TSF片段，与经过SalⅠ和BglⅡ酶切处理的pE1/EGFP载体相连获得pE1/EGFP/AAVS1-TSF。
采用飞捷生物试剂公司的微量基因组DNA极速抽提试剂盒从HEK293细胞中提取人基因组DNA，并以此基因组DNA为模板，用引物P5/P6通过聚合酶链式反应获得上游同源臂AAVS1UP。聚合酶链式反应的条件按照
HS DNA聚合酶试剂盒的说明书进行。用同样的方法以P7和P8为引物，可获得下游同源臂AAVS1DOWN。将AAVS1UP和AAVS1DOWN分别与pGEM-T Easy载体用SolutionⅠ试剂盒（Takara公司购买）连接。将连接产物转化、提取质粒、酶切鉴定并测序获得阳性克隆，分别命名为pGEMT/AAVS1UP和pGEMT/AAVS1DOWN。
将pGEMT/AAVS1UP用SalⅠ和ClaⅠ酶切处理，pGEMT/AAVS1DOWN用ClaⅠ和BglⅡ处理，通过琼脂糖凝胶电泳分别回收AAVS1UP片段和AAVS1DOWN片段，与经过SalⅠ和BglⅡ酶切处理的pE1/EGFP载体用SolutionⅠ试剂盒（Takara公司购买）连接。经过转化、提取质粒、酶切鉴定获得阳性克隆，命名为pE1/EGFP/AAVS11000F。将pE1/EGFP/AAVS11000F用SalⅠ和BglⅡ酶切并回收AAVS11000F片段，与经过XhoⅠ和BglⅡ酶切处理的pE1/EGFP/AAVS1-TSF载体相连，经过转化、提取质粒、酶切鉴定获得阳性克隆，命名为pE1/EGFP/AAVS1-TSF-1000F。用SalⅠ和BglⅡ酶切pE1/EGFP/AAVS1-TSF-1000F，通过琼脂糖凝胶电泳回收AAVS1-TSF-1000F片段，与经过XhoⅠ和BglⅡ酶切处理的pE1/EGFP/AAVS1-TSF-1000F载体用SolutionⅠ试剂盒（Takara公司购买）连接，获得pE1/EGFP/2×AAVS1-TSF-1000F至此获得了携带两个供体DNA片段的打靶载体。将AAVS1-TSF-1000F片段用SolutionⅠ试剂盒（Takara公司购买）连接到XhoⅠ和BglⅡ酶切处理的pE1/EGFP/2×AAVS1-TSF-1000F载体可获得pE1/EGFP/3×AAVS1-TSF-1000F，以此方法可获得pE1/EGFP/4×AAVS1-TSF-1000F、pE1/EGFP/5×AAVS1-TSF-1000F、pE1/EGFP/6×AAVS1-TSF-1000F、pE1/EGFP/7×AAVS1-TSF-1000F、pE1/EGFP/8×AAVS1-TSF-1000F、pE1/EGFP/9×AAVS1-TSF-1000F、pE1/EGFP/10×AAVS1-TSF-1000F。将AAVS1-1000F片段与经过XhoⅠ和BglⅡ酶切处理的pE1/EGFP/AAVS1-1000F载体用SolutionⅠ试剂盒（Takara公司购买）连接，获得对照载体pE1/EGFP/2×AAVS1-1000F，同样的方法可以获得pE1/EGFP/3×AAVS1-1000F、pE1/EGFP/4×AAVS1-1000F、pE1/EGFP/5×AAVS1-1000F、pE1/EGFP/6×AAVS1-1000F、pE1/EGFP/7×AAVS1-1000F、pE1/EGFP/8×AAVS1-1000F、pE1/EGFP/9×AAVS1-1000F、pE1/EGFP/10×AAVS1-1000F。
3、靶向AAVS1位点的基因打靶系统对靶位点的修饰效率检测
以1×106的密度将HEK293细胞接种于A、B、C三个60mm培养皿。次日，通过电转染将质粒导入细胞。电转染条件如下：120V，950μF。转染质粒为：A盘5μg pE1/AAVS1ZFN+20μgpE1/EGFP/AAVS1-1000F;B盘5μg pE1/AAVS1ZFN+20μg pE1/EGFP/AAVS1-TSF-1000F;C盘5μgpE1/AAVS1ZFN+20μg pE1/EGFP/4×AAVS1-TSF-1000F。转染后的第五天收集细胞，用血液/细胞/组织基因组DNA提取试剂盒（天根生化科技有限公司，DP304-02）提取基因组DNA，通过聚合酶链式反应扩增靶位点附近1kb的DNA片段，聚合酶链式反应的条件按照HS DNA聚合酶试剂盒的说明书进行。引物序列如下：
P13:AAVS1detection primer for tgggtcctctccgggcatctct
P14:AAVS1detection primer back gggagttttccacacggacac
取500ng的该聚合酶链式反应的产物，用20U的ClaⅠ酶处理3小时后，跑DNA凝胶电泳检测，用DNA凝胶成像系统拍照。结果如图4。
由图4可见，转染pE1/EGFP/AAVS1-TSF-1000F的HEK293细胞AAVS1位点靶向修饰效率远高于对照组pE1/EGFP/AAVS11000F。而转染pE1/EGFP/4×AAVS1-TSF-1000F的HEK293细胞AAVS1位点的靶向修饰效率相比转染pE1/EGFP/AAVS1-TSF-1000F组进一步提高约3倍。
实施例2
以靶向CCR5位点的基因打靶系统为例，其组成如下：
（1）表达CCR5ZFN的锌指核酸酶表达载体
该表达CCR5ZFN的锌指核酸酶表达载体由两个载体组成，其中一个携带左侧锌指核酸酶CCR5ZFNL，另一个携带右侧锌指核酸酶CCR5ZFNR。该载体以质粒载体pshuttle为基础，在pshuttle的多克隆位点依次引入了真核启动子CMV、靶向CCR5位点的左侧锌指核酸酶CCR5ZFNL或者右侧锌指核酸酶CCR5ZFNR、转录终止信号TKpA，构成表达CCR5ZFN的锌指核酸酶表达载体。该锌指核酸酶表达载体的结构见图5。
CCR5ZFNL的序列见序列表<210>24到gaaatcaact tctag 1035。
CCR5ZFNR的序列见序列表<210>25到ataaactttt ga 1062。
（2）靶向CCR5位点的打靶载体
在pshuttle的多克隆位点携带有串联重复的10个供体DNA片段CCR5-100F，每个供体DNA片段的5＇端和3＇端均插入了CCR5ZFN的识别序列CCR5-TSF，供体DNA片段由上游同源臂CCR5UP、下游同源臂CCR5DOWN和位于两者之间的外源DNA序列（一个碱基G，造成移码突变）组成，上游同源臂是基因组上距离CCR5-TSF1bp长度为50bp的DNA序列，下游同源臂是基因组上距离CCR5-TSF2bp长度为50bp的DNA序列。该靶向CCR5位点的打靶载体命名为10×CCR5-TSF-100F。
CCR5-TSF的序列如下：
Ggtcatcctcatcctgataaactgcaaaagg
CCR5-100F的序列如下：
cctccgctctactcactggtgttcatctttggttttgtgggcaacatgctGtgaagagcatgactgacatctacc tgctcaacctggccatctctgacctg
10×CCR5-TSF-100F的序列见序列表<210>28到ctcgagttag atct1454。
上述靶向CCR5位点的基因打靶系统的构建方法步骤如下：
1、携带CCR5ZFN的锌指核酸酶表达载体的构建
在上海生工合成左侧锌指核酸酶基础骨架，序列见序列表<210>29到gt 722。
合成右侧锌指核酸酶基础骨架，序列见序列表<210>30到ttttgaacta gt 752。
将合成的左侧锌指核酸酶基础结构和右侧锌指核酸酶基础结构分别用ClaⅠ和SpeⅠ酶切处理后，分别与经过同样酶切处理的pUC19/EHL用SolutionⅠ试剂盒（Takara公司购买）连接，将连接产物转化感受态的DH5α细胞，并涂布于含有100μg/ml的氨苄青霉素的LB平板中。挑取菌落接种到含有100μg/ml的氨苄青霉素的LB培养液中，14～16小时后，经碱性裂解法提取质粒DNA，通过酶切鉴定阳性克隆。将所获得阳性克隆分别命名为pUC19/EHL/FokⅠDD、pUC19/EHL/FokⅠRR。
根据已经报道的靶向CCR5位点的锌指蛋白的序列，在上海生工合成靶向CCR5位点的一对锌指蛋白基因，分别命名为CCR5ZFL和CCR5ZFR。并且在CCR5ZFL和CCR5ZFR的两端分别引入了HindⅢ-SalⅠ和KpnⅠ-BglⅡ酶切位点。序列如下：
CCR5ZFL的序列见序列表<210>31到attcatgtcg aca 353。
CCR5ZFR的序列见序列表<210>32到caccaaaatt catagatcta 350。
将CCR5ZFL用HindⅢ和SalⅠ酶切，与经过HindⅢ和XhoⅠ酶切处理的pUC19/EHL/FokⅠDD载体用SolutionⅠ试剂盒（Takara公司购买）连接，经过转化、提取质粒、酶切鉴定，获得阳性克隆命名为pUC19/EHL/CCR5ZFNL。将CCR5ZFR用KpnⅠ和BglⅡ酶切处理，与经过KpnⅠ和BamHⅠ酶切处理的pUC19/EHL/FokⅠRR载体用SolutionⅠ试剂盒（Takara公司购买）连接，经过转化、提取质粒、酶切鉴定，获得阳性克隆命名为pUC19/EHL/CCR5ZFNR。至此获得携带CCR5ZFNL和CCR5ZFNR的锌指核酸酶表达载体。
其它步骤与实施例1相同。
实施例3
以靶向AAVS1位点的基因打靶系统为例，其组成如下：
本实施例的（2）靶向AAVS1位点的打靶载体由以下结构组成，
在pshuttle的多克隆位点携带有串联重复的2个供体DNA片段AAVS1-2000F，每个供体DNA片段的5＇端和3＇端分别插入了AAVS1ZFN的识别序列AAVS1-TSF，供体DNA片段由上游同源臂AAVS1UP、下游同源臂AAVS1DOWN和位于两者之间的外源DNA序列（CMV-eGFP-T2A-luciferase-SV40pA，3000bp片段）组成，上游同源臂是基因组上距AAVS1-TSF3000bp的长度为1000bp的DNA序列，下游同源臂是基因组上距离AAVS1-TSF100bp的长度为1000bp的DNA序列。
AAVS1-2000F的序列见序列表<210>33到gctgggttgg agtgg 5385。
其它结构与实施例1相同。
实施例4
以靶向SNCA位点的基因打靶系统为例，其组成如下：
（1）表达AAVS1ZFN的锌指核酸酶表达载体
在腺病毒载体pAdEasy-1的E1区携带有锌指核酸酶表达原件CMV-SNCA ZFNL-F2A-SNCAZFNR-SV40pA，由CMV启动子同时表达左侧锌指核酸酶SNCA ZFNL和右侧锌指核酸酶SNCAZFNR，左侧锌指核酸酶SNCA ZFNL与右侧锌指核酸酶SNCA ZFNR之间通过自剪切多肽F2A相连。表达SNCA ZFN的锌指核酸酶表达载体的结构见图6。本实施例的腺病毒载体也可以用非整合型慢病毒载体、腺相关病毒载体、杆状病毒载体中的任意一种替换。
SNCA ZFNL-F2A-SNCA ZFNR的序列见序列表<210>34到ataaactttt ga 2472。
（2）靶向SNCA位点的打靶载体
在腺病毒载体pAdeasy-1的E1区携带有串联重复的2个供体DNA片段SNCA-3500F，每个供体DNA片段的5＇端和3＇端均插入了SNCA ZFN的识别序列SNCA-TSF，供体DNA片段由上游同源臂SNCA UP、下游同源臂SNCA DOWN和位于两者之间的外源DNA序列（loxp序列）组成，上游同源臂是基因组上距SNCA-TSF1bp的长度为3000bp的DNA序列，下游同源臂是基因组上距离SNCA-TSF1bp的长度为500bp的DNA序列，靶向SNCA位点的打靶载体的结构见图7。本实施例的腺病毒载体也可以用非整合型慢病毒载体、腺相关病毒载体、杆状病毒载体中的任意一种替换。
SNCA-TSF的序列如下：
GGAGTGGTGCATGGTGTGGCAACAGGTAAGCTCCATTGTGC
SNCA-3500F的序列见序列表<210>36到attttaggct gggc 3534。
Claims (5)
1.一种基因打靶系统，由位点特异性切割核酸酶表达载体和打靶载体两部分组成，其特征在于：所述的打靶载体包含2～10个供体DNA片段，每个供体DNA片段的5＇端和3＇端分别插入位点特异性切割核酸酶的识别序列，供体DNA由上游同源臂、下游同源臂和位于两者之间的外源DNA序列组成；
上述的位点特异性切割核酸酶表达载体是携带锌指核酸酶的表达载体、转录激活子样效应因子核酸酶的表达载体、RNA介导的核酸酶RNA:Cas9的表达载体中的任意一种。
2.根据权利要求1所述的基因打靶系统，其特征在于：所述的位点特异性切割核酸酶的识别序列是基因组上位点特异性切割核酸酶结合的长度为20bp～50bp的DNA序列。
3.根据权利要求1所述的基因打靶系统，其特征在于：所述的外源DNA序列是长度为1bp～3000bp的DNA片段。
4.根据权利要求1所述的基因打靶系统，其特征在于：所述的上游同源臂、下游同源臂是分别与位点特异性切割核酸酶识别位点上游和下游的部分基因组序列同源的两段长度为50bp～3000bp的DNA片段。
5.根据权利要求4所述的基因打靶系统，其特征在于：所述的部分基因组序列是距离锌指核酸酶识别位点1bp～3000bp的DNA序列。
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