CN105802980A - Gateway兼容性CRISPR/Cas9系统及其应用 - Google Patents
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Abstract
本发明公布了一种Gateway兼容性CRISPR/Cas9系统及其应用。所述CRISPR/Cas9系统包括中间载体和终载体,其中:中间载体包含Ⅲ型启动子负责驱动的sgRNA作用元件,靠近sgRNA作用元件序列5’端位置含有靶点序列连入位点,在Ⅲ型启动子负责驱动的sgRNA作用元件的整体序列两侧分别含有重组位点attL1和attL2;终载体包含Ⅱ型启动子负责驱动的Cas9基因,同时含有重组位点attR1和attR2,在attR1和attR2之间含有反向选择性标记基因。中间载体和终载体通过Gateway LR反应生成含sgRNA作用元件和Cas9基因的表达载体,可广泛应用在生物体的基因组编辑中。
Description
技术领域
本发明涉及植物基因工程领域,具体涉及一种Gateway兼容性CRISPR/Cas9系统及其在植物基因组编辑中的应用。
背景技术
CRISPR/Cas9系统是新兴的准确高效的基因编辑系统,该系统包括两个基本元件sgRNA和Cas9基因,前者由RNA聚合酶Ⅲ类启动子负责驱动转录出一个短的单链引导RNA(sgRNA)。后者是由RNA聚合酶Ⅱ类组成型启动子负责转录表达mRNA,进而翻译生成Cas9核酸酶。在体内,sgRNA和Cas9形成功能复合物,sgRNA中含有基因组中位点特异性的靶点序列,负责引导复合物准确结合到基因组特异位置,Cas9核酸酶负责将此处DNA双链切割形成平末端双链断裂,生物体启动DNA损伤修复机制,由于修复的不精确性往往导致靶点处基因产生突变,导致其丧失功能。
目前常规的CRISPR/Cas9系统包括两个载体,一个是包含Ⅲ型启动子负责驱动的sgRNA的中间载体,一个是包含Ⅱ型启动子负责驱动的Cas9基因的表达载体。然后通过酶切连接的方法,将Ⅲ型启动子负责驱动的sgRNA元件整合到含有Cas9的表达载体中,生成最终的包含sgRNA和Cas9两个元件的表达载体(见图1)。该常规方法在操作中需要经过酶切、胶回收和片段连接等三个过程,耗时较长而且相对费力。此外,如果sgRNA中的靶点含有后续酶切反应所用的酶切位点,则会导致实验失败。
发明内容
针对目前常规CRISPR/Cas9系统在操作中存在的费时费力以及靶点中可能含有后续所需酶切位点等问题,本发明提供了一种Gateway兼容性CRISPR/Cas9系统,以更为方便快捷地用于基因组编辑。
本发明提供的CRISPR/Cas9系统包括中间载体和终载体,其中:所述中间载体包含Ⅲ型启动子负责驱动的sgRNA作用元件,在sgRNA作用元件序列内靠近5’端位置含有靶点序列连入位点,Ⅲ型启动子负责驱动的sgRNA作用元件的整体序列两侧分别含有重组位点attL1和attL2;所述终载体包含Ⅱ型启动子负责驱动的Cas9基因,同时含有重组位点attR1和attR2,在attR1和attR2之间含有反向选择性标记基因(counter-selectablemarkergene)。
在本发明中,术语“Ⅱ型启动子”是指RNA聚合酶Ⅱ类启动子,术语“Ⅲ型启动子”是指RNA聚合酶Ⅲ类启动子。
所述Ⅱ型启动子例如p35S、pDD45和pYAO等启动子,所述Ⅲ型启动子例如U6、H1等启动子。
所述“反向选择性标记基因”例如自杀基因ccdB。
sgRNA元件序列内靠近5’端位置含有靶点序列连入所需位点,通常是个多克隆位点,可以包含两个或更多个限制性内切酶的酶切位点,以便根据实际应用的需要,在此连入位点通过酶切连接方法连入靶点序列。
在终载体上,相关序列按attR1、反向选择性标记基因、attR2和Ⅱ型启动子负责驱动的Cas9基因的顺序排列。
本发明的CRISPR/Cas9系统具有Gateway兼容性,在进行基因组编辑的应用中,首先通过酶切连接等方法将靶向基因组中目标基因的靶点序列连入中间载体的sgRNA作用元件中,然后通过GatewayLR反应将中间载体所含的Ⅲ型启动子负责驱动的sgRNA作用元件的区域转移到终载体上,生成最终的含有Ⅲ型启动子负责驱动的sgRNA作用元件和Ⅱ型启动子负责驱动的Cas9基因的表达载体。在生成的表达载体中,Ⅲ型启动子负责驱动的sgRNA作用元件的整体序列位于LR反应生成的新重组位点attB1和attB2之间。利用该表达载体转化生物体,得到目标基因被编辑的生物体。
图2显示了本发明的一个具体例子,中间载体包含拟南芥Ⅲ型启动子pAtU6-26负责驱动的sgRNA作用元件,在该区域的两侧分别是重组位点attL1和attL2;终载体包含Ⅱ型启动子(Pro)负责驱动的Cas9基因,以及选择标记基因CmR和自杀基因ccdD,在选择标记基因CmR和自杀基因ccdD两侧分别是重组位点attR1和attR2;中间载体和终载体通过GatewayLR反应生成含有sgRNA和Cas9的表达载体。
应用基因重组技术可以通过很多途径构建本发明的CRISPR/Cas9系统,本发明在具体实施方式中描述了一种途径,但本领域的技术人员应该理解,本发明的CRISPR/Cas9系统的构建不应局限于该途径。
本发明具体实施方式中描述的一种具有Gateway兼容性的CRISPR/Cas9系统构建方法,包括以下步骤:
1)将包含Ⅲ型启动子负责驱动的sgRNA作用元件的片段克隆到Gateway系统的入门载体(Entryvector)中,得到本发明的中间载体;
2)将包含Ⅱ型启动子负责驱动的Cas9基因的片段克隆到Gateway系统的目的载体(Destinationvector)中,得到本发明的终载体。
在本发明的实施例中,上述步骤1)是将包含Ⅲ型启动子负责驱动的sgRNA作用元件的片段通过pENTR/D-TOPO克隆系统进行TOPO反应生成中间载体。
在本发明的实施例中,上述步骤2)首先通过PCR克隆或限制性内切酶酶切等方法从基因组或已有载体获得一种特定Ⅱ型启动子片段和Cas9基因片段,然后通过酶切连接的方法将两者连入含有重组位点attL3和attL4的Gateway克隆系统兼容的辅助中间载体中,在此载体中Ⅱ型启动子和Cas9基因整体片段位于attL3和attL4之间;然后将此辅助中间载体与含有attR3和attR4重组位点的Gateway克隆系统目的载体进行LR反应,生成含有Ⅱ型启动子驱动的Cas9基因的表达载体;最后,通过PCR克隆或其他方法获得attR1-反向选择性标记基因-attR2片段,通过限制性内切酶酶切方法将此片段连入上述表达载体中Ⅱ型启动子的5’端,从而得到本发明的终载体。
本发明所提供的Gateway兼容性的CRISPR/Cas9系统在进行基因组编辑时,相对于传统方法具有操作简单、步骤较少、节省时间、节约成本以及无需考虑靶点中含有的酶切位点等优点。
附图说明
图1.常规CRISPR/Cas9系统载体及操作流程,其中RE1和RE2分别表示酶切位点1和酶切位点2。
图2.本发明Gateway兼容性CRISPR/Cas9系统载体及操作流程。
图3.本发明实施例用到的辅助载体pAtU6-26:sgRNA-pBluescript、p35S:Cas9-pBluescript和pYAO:Cas9-pCambia1300的结构示意图。
图4.Gateway克隆系统兼容性CRISPR/Cas9中间载体构建流程图。
图5.L3L4-MCS-pENTR辅助中间载体构建流程图。
图6.p35S:Cas9-pCambia1300和pDD45:Cas9-pCambia1300载体构建流程图。
图7.Gateway克隆系统兼容性CRISPR/Cas9终载体构建流程图。
图8.基于本发明的CRISPR/Cas9表达载体构建流程。
图9.T1代转基因阳性植株靶向基因突变示例,图中L1、L2、L3表示三个遗传独立的T1代转基因植株。
具体实施方式
下面结合附图,通过实施例详细介绍本发明Gateway兼容性CRISPR/Cas9系统的中间载体和终载体具体构建步骤,并通过实施例进一步阐述本发明的使用方法和实验结果。
1载体改造所需的9个基础载体:
(1)pAtU6-26:sgRNA-pBluescript:在载体pBluescript的KpnI/XhoI位点引入pAtU6-26:sgRNA片段(SEQIDNo:1),本载体由中国科学院上海植物逆境生物学研究中心朱健康教授实验室赠送,载体详细信息可参考相关文献1:Z.,etal.,EfficientgenomeeditinginplantsusingaCRISPR/Cassystem.CellRes,2013.23(10):p.1229-32.,见图3中(a);
(2)p35S:Cas9-pBluescript:在载体pBluescript的KpnI/EcoRI位点引入35S:Cas9片段(SEQIDNo:2),本载体由中国科学院上海植物逆境生物学研究中心朱健康教授实验室赠送,载体详细信息可参考相关文献1:Z.,etal.,EfficientgenomeeditinginplantsusingaCRISPR/Cassystem.CellRes,2013.23(10):p.1229-32.,见图3中(b);
(3)pYAO:Cas9-pCambia1300:在载体pCambia1300的KpnI/EcoRI位点引入pYAO:Cas9片段(SEQIDNo:3),本载体由中国科学院遗传与发育生物学研究所谢旗研究员实验室构建和赠送,载体详细信息可参考相关文献2:Yan,L.,etal.,High-EfficiencyGenomeEditinginArabidopsisUsingYAOPromoter-DrivenCRISPR/Cas9System.MolPlant,2015.8(12):p.1820-3.,见图3中(c);
(4)pBluescript载体;
(5)Gateway克隆系统终载体(Invitrogen):pDONR221、pK2WG7、pK7m34GW、pB7m34GW和pH7m34GW等。
2Gateway克隆系统兼容性CRISPR/Cas9系统的构建
在此构建的Gateway克隆系统兼容性CRISPR/Cas9系统包括1种中间载体和9种终载体,下面将详细介绍这些载体的构建过程。
2.1Gateway克隆系统兼容性CRISPR/Cas9中间载体的构建
首先以pAtU6-26:sgRNA-pBluescript为模板,利用PCR方法将pAtU6-26:sgRNA片段进行扩增。所用引物如下:
正向引物pAtU6-26sg-topo-FP:5’-caccGGTACCACTAGTAGCTTCGTTGAACAAC-3’(SEQIDNo:4);
反向引物pAtU6-26sg-topo-RP:5’-GAGCTCGTCGACAAGCTTTCTAGACGGCCGCCAGTGTGATGGATATC-3’(SEQIDNo:5)。
PCR产物经琼脂糖凝胶电泳鉴定并回收,回收产物通过pENTR/D-TOPO克隆系统进行TOPO反应生成pAtU6-26:sgRNA-pENTR中间载体。由于将来靶点序列需要通过连接反应连入pAtU6-26:sgRNA片段中的BbsI位点,而在中间载体的骨架中存在一个BbsI位点,所以需要将pAtU6-26:sgRNA-pENTR骨架中的BbsI位点进行点突变进行消除。在此以pAtU6-26:sgRNA-pENTR为模板,利用PCR方法进行点突变,所用引物如下:
正向引物pENTR-backbone-bbs1-mut-FP:5’-GGCCCAGAGTTCCGACTGAGCCTTTCGTTTTATTTGAT-3’(SEQIDNo:6);
反向引物pENTR-backbone-bbs1-mut-RP:5’-CTCAGTCGGAACTCTGGGCCTTTCGTTTTATCTGTTG-3’(SEQIDNo:7)。
PCR产物经琼脂糖凝胶电泳鉴定并回收,将回收产物转化DH5α大肠杆菌感受态,通过测序筛选阳性克隆,扩繁阳性菌落,最终得到骨架中BbsI位点完成点突变的pAtU6-26:sgRNA-pENTR(见图4)。
2.2Gateway克隆系统兼容性CRISPR/Cas9终载体的构建
本方法构建的Gateway克隆系统兼容性CRISPR/Cas9终载体包括3种不同的用于驱动Cas9基因表达的启动子,分别是p35S、pDD45和pYAO,以及3种不同植物抗性筛选标记基因,分别是Kan、Bar和Hyg,两两组合,共9种终载体,下面将详细介绍这些终载体的构建过程。
2.2.1L3L4-MCS-pENTR辅助中间载体的构建
首先以pBluescript为模板,利用PCR扩增载体中的多克隆位点(MCS)片段,正向引物pBS-mcs-bp-FP:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGCTACCGGGCCCCCCCTCG-3’(SEQIDNo:8),反向引物pBS-mcs-bp-RP:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTATCTGAGCTCCACCGCGGTGG-3’(SEQIDNo:9),将PCR产物经琼脂糖凝胶电泳鉴定并回收,回收产物与Gateway克隆系统的pDONR221进行BP反应,生成L1L2-MCS-pENTR中间载体。载体MCS位点中的KpnI位点也已在此步骤中突变去除。
然后以L1L2-MCS-pENTR为模板,利用PCR方法将此载体中的attL1和attL2位点分别突变为attL3和attL4位点,将attL1突变为attL3所用的正向引物attL1toL3-mut-FP:5’-ACTTTGTATAATAAAGTTGGCTTAGCTACCGGGC-3’(SEQIDNo:10),反向引物attL1toL3-mut-RP:5’-AACTTTATTATACAAAGTTGGCATTATAAA-3’(SEQIDNo:11);将attL2突变为attL4所用的正向引物attL2toL4-mut-FP:5’-tggagctcagataccCAACTTTTCTATACAAAGTTGGCATTAT-3’(SEQIDNo:12),反向引物attL2toL4-mut-RP:5’-ACTTTGTATAGAAAAGTTGGGTATCTGAGCTCCACCGCGG-3’(SEQIDNo:13),最终生成L3L4-MCS-pENTR辅助中间载体(见图5)。
2.2.2pDD45:Cas9-pBluescript载体的构建
以拟南芥基因组DNA为模板,利用PCR方法扩增DD45基因(AGI,AT2G21740)启动子,正向引物pDD45-kpn1-FP:5’-GGGGTACCAAATGTTCCTCGCTGACGTA-3’(SEQIDNo:14),反向引物pDD45-xho1-RP:5’-CCGCTCGAGCATTATTCTTTCTTTTTGGGGTTTTTGTTTTG-3’(SEQIDNo:15),扩增产物经琼脂糖凝胶电泳鉴定并回收。正向引物和反向引物的5’端分别含有KpnI和XhoI酶切位点。利用KpnI和XhoI限制性内切酶酶切扩增产物,酶切产物经琼脂糖凝胶电泳鉴定并回收,回收产物命名为pDD45-KX片段。
利用KpnI和XhoI限制性内切酶酶切p35S:Cas9-pBluescript载体产生35S启动子和Cas9-pBluescript骨架两个片段,经琼脂糖凝胶电泳鉴定并回收Cas9-pBluescript骨架,回收产物命名为Cas9-pBluescript-KX。然后利用T4连接酶分别将pDD45-KX与Cas9-pBluescript-KX进行连接反应,生成pDD45:Cas9-pBluescript载体(参见图6)。
2.2.3p35S:Cas9-pCambia1300和pDD45:Cas9-pCambia1300载体的构建
利用KpnI和EcoRI限制性内切酶分别酶切p35S:Cas9-pBluescript和pDD45:Cas9-pBluescript载体,酶切所得p35S:Cas9和pDD45:Cas9片段经琼脂糖凝胶电泳鉴定并回收,回收产物分别命名为p35S:Cas9-KE和pDD45:Cas9-KE片段。利用KpnI和EcoRI限制性内切酶酶切pYAO:Cas9-pCambia1300质粒,酶切所得pCambia1300骨架片段经琼脂糖凝胶电泳鉴定并回收,回收产物命名为pCambia1300-KE片段。然后利用T4连接酶将p35S:Cas9-KE和pDD45:Cas9-KE分别与pCambia1300-KE进行连接反应,生成p35S:Cas9-pCambia1300和pDD45:Cas9-pCambia1300载体。
至此,本系统已包括三种不同启动子驱动Cas9的pCambia1300载体,分别是p35S:Cas9-pCambia1300、pDD45:Cas9-pCambia1300和pYAO:Cas9-pCambia1300,在此将这三种载体统称为Pro:Cas9-pCombia1300载体(见图6)。
2.2.4Pro:Cas9-L3L4-pENTR中间载体构建
利用SpeI和EcoRI限制性内切酶分别对三种Pro:Cas9-pCombia1300载体进行酶切,酶切所得Pro:Cas9片段经琼脂糖凝胶电泳鉴定并回收,回收产物命名为Pro:Cas9-SE片段。同时利用SpeI和EcoRI限制性内切酶酶切L3L4-MCS-pENTR质粒,酶切产物经琼脂糖凝胶电泳鉴定并回收,回收产物命名为L3L4-MCS-pENTR-SE片段。然后利用T4连接酶将Pro:Cas9-SE和L3L4-MCS-pENTR-SE进行连接反应,生成Pro:Cas9-L3L4-pENTR中间载体(见图7)。
2.2.5Pro:Cas9-pEXP表达载体的生成
将三种不同的Pro:Cas9-L3L4-pENTR载体分别与三种含有不同植物抗性筛选基因的Gateway终载体pDEST,分别为pK7m34GW、pB7m34GW和pH7m34GW,进行Gateway克隆系统LR反应分别生成Pro:Cas9-pK7m34GW-pEXP、Pro:Cas9:Cas9-pB7m34GW-pEXP和Pro:Cas9:Cas9-pH7m34GW-pEXP9种表达载体,在此将9种载体统称为Pro:Cas9-pEXP表达载体(见图7)。
2.2.6Pro:Cas9-R1R2-pDEST终载体构建
利用KpnI和SpeI限制性内切酶分别9种Pro:Cas9-pEXP表达载体进行酶切,酶切产物经琼脂糖凝胶电泳鉴定并回收,统称为Pro:Cas9-pEXP-SK。同时以Gateway终载体pK2WG7为模板,利用PCR扩增载体中的p35S-attR1-CmR-ccdB-attR2-T35S片段,正向引物p35S-FP:5’-AGATGCCTCTGCCGACAGTGGT-3’(SEQIDNo:16),反向引物T35S-Kpn1-RP:5’-ggggtaccAGGTCACTGGATTTTGGTTTTAGG-3’(SEQIDNo:17),扩增产物经琼脂糖凝胶电泳鉴定并回收。attR1序列和反向引物的5’端分别含有SpeI和KpnI酶切位点。利用KpnI和SpeI限制性内切酶酶切p35S-attR1-CmR-ccdB-attR2-T35S,酶切产物经琼脂糖凝胶电泳鉴定并回收,产物命名为attR1-CmR-ccdB-attR2-T35S-SK。然后利用T4连接酶将attR1-CmR-ccdB-attR2-T35S-SK分别与9种Pro:Cas9-pEXP-SK与进行连接反应,生成9种Pro:Cas9-R1R2-pDEST终载体(见图7)。
至此本Gateway克隆系统兼容性CRISPR/Cas9基因编辑系统所有载体改造完成,包括1个中间载体和9个Pro:Cas9-R1R2-pDEST终载体(见表1)。
表1.Gateway克隆系统兼容性CRISPR/Cas9基因编辑系统载体信息
表1中载体简称一列K表示植物抗性筛选Kan基因;B表示植物抗性筛选Bar基因;H表示植物抗性筛选Hyg基因;2表示2×35S启动子;D表示DD45基因启动子;Y表示YAO基因启动子;C表示Cas9基因;GW表示Gateway兼容性。
基于本发明的CRISPR/Cas9表达载体构建流程
通过酶切连接方法,将靶点序列连入2.1中构建的pAtU6-26:sgRNA-pENTR载体的BbsI位点,生成含有靶点序列的pAtU6-26:sgRNA-pENTR2中间载体,然后将该载体与2.2中构建的Pro:Cas9-R1R2-pDEST终载体进行Gateway克隆系统LR反应,生成最终的Pro:Cas9-pAtU6-26:sgRNA-pEXP表达载体(见图8)。将表达载体转化农杆菌GV1301感受态,然后将农杆菌侵染植物,T0代植物的种子铺在含有相应植物筛选抗生素的1/2MS培养基上进行筛选,最终获得转基因阳性植物。
3.植物基因组编辑试验
本例以一个测试靶点同时靶向拟南芥中三个同源基因(Gene-1、Gene-2和Gene-3)以检测本系统的可行性及工作效率。所选靶点序列的正向引物为Guide-test-FP:5’-GATTGCGCAAGAGCTTGTATGAGA-3’(SEQIDNo:18),反向引物为Guide-test-RP:5’-AAACTTCAATCCAGTAGCAAGTCC-3’(SEQIDNo:19),所选终载体为pBYCGW。选取20株T1代转基因阳性植株,对每株植物提取DNA,在靶点序列两侧设计引物并进行PCR扩增,对每组扩增产物进行测序,然后将测序结果与原始基因序列进行序列比对,以检测靶点处是否发生突变。结果显示,在检测的20株植物中,14株植物(70%)的Gene-1发生突变,18株植物(90%)的Gene-2发生突变,19株植物(94.5%)的Gene-3发生突变。本例给出3个独立的T1代转基因阳性植株(L1、L2和L3)中Gene-1、Gene-2和Gene-3三个基因靶点处的突变情况(见图9)。这些结果显示本系统能够正常工作并显示较高的工作效率。
Claims (9)
1.一种CRISPR/Cas9系统,包括中间载体和终载体,其中:所述中间载体包含Ⅲ型启动子负责驱动的sgRNA作用元件,在sgRNA作用元件序列内靠近5’端位置含有靶点序列连入位点,在Ⅲ型启动子负责驱动的sgRNA作用元件的整体序列两侧分别含有重组位点attL1和attL2;所述终载体包含Ⅱ型启动子负责驱动的Cas9基因,同时含有重组位点attR1和attR2,在attR1和attR2之间含有反向选择性标记基因。
2.如权利要求1所述的CRISPR/Cas9系统,其特征在于,所述Ⅱ型启动子选自p35S、pDD45和pYAO启动子中的一种;所述Ⅲ型启动子是U6启动子或H1启动子。
3.如权利要求1所述的CRISPR/Cas9系统,其特征在于,所述反向选择性标记基因为自杀基因ccdB。
4.如权利要求1所述的CRISPR/Cas9系统,其特征在于,所述靶点序列连入位点是包含两个或更多个限制性内切酶酶切位点的多克隆位点。
5.如权利要求1所述的CRISPR/Cas9系统,其特征在于,在所述终载体上,相关序列按ttR1、反向选择性标记基因、attR2和Ⅱ型启动子负责驱动的Cas9基因的顺序排列。
6.权利要求1~5任一所述CRISPR/Cas9系统在基因组编辑中的应用,首先将靶向基因组中目标基因的靶点序列连入中间载体的靶点序列连入位点中,然后通过GatewayLR反应将中间载体所含的Ⅲ型启动子负责驱动的sgRNA作用元件的区域转移到终载体上,生成含有Ⅲ型启动子负责驱动的sgRNA作用元件和Ⅱ型启动子负责驱动的Cas9基因的表达载体,利用该表达载体转化生物体,得到目标基因被编辑的生物体。
7.权利要求1~5任一所述CRISPR/Cas9系统的制备方法,包括以下步骤:
1)将包含Ⅲ型启动子负责驱动的sgRNA作用元件的片段克隆到Gateway系统的入门载体中,得到所述中间载体;
2)将包含Ⅱ型启动子负责驱动的Cas9基因的片段克隆到Gateway系统的目的载体中,得到所述终载体。
8.如权利要求7所述的制备方法,其特征在于,步骤1)将包含Ⅲ型启动子负责驱动的sgRNA作用元件的片段通过pENTR/D-TOPO克隆系统进行TOPO反应生成中间载体。
9.如权利要求7所述的制备方法,其特征在于,步骤2)首先获得一种Ⅱ型启动子片段和Cas9基因片段,将两者连入含有重组位点attL3和attL4的Gateway克隆系统兼容的辅助中间载体中,并使Ⅱ型启动子和Cas9基因整体片段位于attL3和attL4之间;然后将此辅助中间载体与含有attR3和attR4重组位点的Gateway克隆系统目的载体进行LR反应,生成含有Ⅱ型启动子驱动的Cas9基因的表达载体;接着获得attR1-反向选择性标记基因-attR2片段,将此片段连入含有Ⅱ型启动子驱动的Cas9基因的表达载体中,位于Ⅱ型启动子的5’端,从而得到所述终载体。
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