CN104004778A - 含有CRISPR/Cas9系统的靶向敲除载体及其腺病毒和应用 - Google Patents
含有CRISPR/Cas9系统的靶向敲除载体及其腺病毒和应用 Download PDFInfo
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Abstract
本发明公开了一种含有CRISPR/Cas9系统的靶向敲除载体及其腺病毒和应用，该靶向敲除载体由pX330-U6-Chimeric_BB-CBh-hSpCas9质粒用EcoRI和SacII酶切、补平后连接经BstXI酶切、补平的pAdTrack-CMV质粒，然后用BbsI线性化后连入目的基因的特异靶序列，再用PmeⅠ线性化和CIAP碱性磷酸酶去磷酸化后与pAdEasy-1质粒重组而得，获得的靶向敲除载体能够在靶序列区域突变基因序列，且突变率高，达30.6％-45.8％，可以用于基因定点突变，为基因治疗奠定了基础。
Description
技术领域
本发明属于表达系统领域，具体涉及含有CRISPR/Cas9系统的靶向敲除载体及其腺病毒和应用。
背景技术
CRISPR/Cas9系统是一种存在于细菌和古细菌中的一种后天免疫系统，以消灭外来的核酸或噬菌体，并在自身基因组中CRISPR位点留下能表达与入侵病毒基因组序列相匹配的小分子RNA，用以保护细菌和古细菌不受病毒的侵害。当含有CRISPR/Cas9系统的细菌和古细菌感染病毒后，CRISPR RNA就能通过互补序列结合病毒基因组，并表达CRISPR相关酶，由于CRISPR相关酶属于核酸酶，能切割病毒DNA分子，从而阻止病毒复制。自2013年以后，研究者们在《Science》、《Nature Biotechnology》等杂志上发表多篇文章介绍CRISPR/Cas9系统，并且已成功在人类、小鼠、斑马鱼等物种上实现精确的基因修饰。但是，目前CRISPR/Cas9相关的实验对象都局限在细胞水平的操作，例如，细胞系或者单细胞胚胎等，并未将CRISPR/Cas9基因靶向操作技术直接用于活体动物。
腺病毒载体法是基因治疗中最有前途的基因转移方法之一。载体容易构建和操作，宿主范围广，感染性强，腺病毒载体能有效地将外源基因转移到各种靶细胞或组织中。可经不同途径进入不同组织，能感染分化后的非分裂期细胞，并且腺病毒基因不整合到宿主细胞中，无插入突变激活癌基因的危险，外源基因能游离地表达。近年，腺病毒载体引起了科研人员的关注，广泛用于遗传病、传染病和肿瘤等疾病的基因治疗的研究和应用。但是并未用于某些基因进行准确的修饰。如：镰刀形人类遗传病的镰刀形贫血症，是由于血红素S的异常造成的。正常表达血红素S蛋白基因的一个碱基T(胸腺嘧啶)突变为A(鸟嘌呤)，导致血红素S蛋白中的一个谷氨酸突变为缬氨酸。红血球由正常的圆盘状的细胞变异成新月形细胞的一种遗传性疾病。患者会因为红血球功能异常及坏损而导致血液循环不良及剧烈疼痛。我们发明的腺病毒介导的CRISPR/Cas9基因靶向操作系统可以真对动物体的基因组进行靶向修饰操作，那么对于像人类由于基因突变带来的疾病的治疗，例如肿瘤、镰刀形贫血症、白化病等，将会获得突破性的进展。
发明内容
有鉴于此，本发明的目的之一在于提供含有CRISPR/Cas9系统的靶向敲除载体；本发明的目的之二在于提供含靶向敲除载体的腺病毒；本发明的目的之三在于提供靶向敲除载体的在制备诱导基因突变试剂中的应用；本发明的目的之四在于提供靶向敲除载体在制备肿瘤抑制剂中的应用。
1、含有CRISPR/Cas9系统的靶向敲除载体，所述靶向敲除载体由pX330-U6-Chimeric_BB-CBh-hSpCas9质粒用EcoRI和SacII酶切、补平后连接经BstXI酶切、补平的pAdTrack-CMV质粒，然后用BbsI线性化后连入目的基因的特异靶序列，再用PmeⅠ线性化和CIAP碱性磷酸酶去磷酸化后与pAdEasy-1质粒重组而得。
优选的，所述目的基因为GFP或c-myc基因。
优选的，目的基因为GFP的特异靶序列如SEQ ID NO.1、SEQ ID NO.4或SEQ ID NO.7所示。
优选的，目的基因为c-myc基因的特异靶序列如SEQ ID NO.12、SEQ ID NO.15或SEQ IDNO.18所示。其中作用于SEQ ID NO.12所示特异靶序列的核苷酸为SEQ ID NO.13和SEQ IDNO.14退火形成的双链，作用于SEQ ID NO.15所示靶序列的核苷酸序列为SEQ ID NO.16和SEQ ID NO.17脱火形成的双链，作用于SEQ ID NO.18所示靶序列的核苷酸序列为SEQ IDNO.19和SEQ ID NO.20退火形成的双链。
更优选的，所述特异靶序列如SEQ ID NO.12所示。
2、含有所述靶向敲除载体的腺病毒。
3、所述靶向敲除载体在制备突变特异靶序列的试剂中的应用。
4、所述靶向敲除载体在制备抑制肿瘤的试剂中的应用。
本发明的有益效果在于：本发明公开了一种含有CRISPR/Cas9系统的靶向敲除载体及其腺病毒和应用，该靶向敲除载体能够在靶序列区域突变基因序列，且敲除率高，达30.6％-45.8％，因此该靶向敲除载体能够用于基因定点突变，可以用于基因治疗，为临床上基因治疗疾病奠定了基础。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图：
图1为转GFP小鼠PCR产物经T7Endonuclease I酶切电泳检测结果图。
图2为流式细胞仪分析小鼠各组织的细胞结果图(A：感染Ad-pSpCas9-T2病毒液小鼠；B：感染Ad-pSpCas9(BB)病毒液小鼠；C：感染生理盐水小鼠)。
图3为感染Ad-pSpCas9-T2病毒液的转GFP小鼠的GFP基因扩增产物测序结果(“-”代表丢失碱基，“+”代表插入碱基)。
图4为转c-myc基因小鼠PCR产物经T7Endonuclease I酶切电泳检测结果图。
图5为感染Ad-pSpCas9-T4病毒液的转c-myc小鼠的c-myc基因扩增产物测序结果(“-”代表丢失碱基，“+”代表插入碱基)。
图6为转c-myc基因小鼠尾静脉注射腺病毒存活统计结果。
具体实施方式
下面将结合附图，对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(第三版，J.萨姆布鲁克等著)中所述的条件，或按照制造厂商所建议的条件。
实施例1
1、构建Crispr/Cas9腺病毒载体
(1)将pX330-U6-Chimeric_BB-CBh-hSpCas9质粒(Addgene plasmid ID:42230)用EcoRI和SacII酶切，酶切后产物经1％的琼脂糖电泳，回收含有hSpCas9的片段，并命名为U6-Chimeric_BB-CBh-hSpCas9。
(2)将pAdTrack-CMV腺病毒载体用BstXI进行酶切去除CMV启动子及GFP片段，酶切后产物经1％的琼脂糖电泳，回收载体骨架，并命名为pAdTrack载体骨架。
(3)将步骤(1)酶切所得回收产物和步骤(2)所得载体骨架用klenow酶补平，然后用T4连接酶在16℃条件下过夜连接，得重组载体，命名为pAdTrack-U6-Chimeric_BB-CBh-hSpCas9。
将获得的重组载体pAdTrack-U6-Chimeric_BB-CBh-hSpCas9转化大肠杆菌DH5α感受态，并涂布于Kan浓度为100μg/mL的固体LB平板中，37℃倒置培养过夜。挑取生长良好的单克隆，于15mL Kan浓度为100μg/mL的LB液体培养基中，37℃摇菌过夜，小量质粒提取，提取质粒通过琼脂糖凝胶电泳，检测大小正确的载体。然后将大小正确的重组载体pAdTrack-U6-Chimeric_BB-CBh-hSpCas9送上海生工生物工程有限公司测序。把测序正确的crispr/cas9腺病毒载体命名为pAdTrack-pSpCas9(BB)。
2、构建敲除GFP基因Crispr/Cas9腺病毒载体
(1)将腺病毒载体pAdTrack-pSpCas9(BB)用BbsI酶切，回收线性片段。
(2)利用在线工具ZiFiT Targeter version4.0设计针对GFP基因的3个靶点的寡核苷酸，具体为：
第一个靶点为T1：5’-ccgcgccgaggtgaagttcg-3’(SEQ ID NO.1)；设计的寡核苷酸对为：5’-caccgccgcgccgaggtgaagttcg-3’(SEQ ID NO.2)；5’-aaaccgaacttcacctcggcgcggc-3’(SEQID NO.3)；
第二个靶点为T2，5’-gtgaaccgcatcgagctgaa-3’(SEQ ID NO.4)；设计的寡核苷酸对为：5’-caccgtgaaccgcatcgagctgaa-3’(SEQ ID NO.5)；5’-aaacttcagctcgatgcggttcac-3’(SEQ IDNO.6)；
第三个靶点为T3，5’-aggaggacggcaacatcctg-3’(SEQ ID NO.7)；设计的寡核苷酸对为：5’-caccgaggaggacggcaacatcctg-3’(SEQ ID NO.8)；5’-aaaccaggatgttgccgtcctcctc-3’(SEQID NO.9)。
(3)将3对寡核苷酸分别加热至95℃后缓慢降至室温退火，形成双链，然后分别连入步骤(1)酶切后的pAdTrack-pSpCas9(BB)线性片段，将连接产物、转化大肠杆菌DH5α感受态细胞，并涂布于Kan浓度为100μg/mL的LB固体平板上培养过夜，挑取生长良好的单克隆，于15mL Kan浓度为100μg/mL的LB液体培养基中，37℃摇菌过夜，提取质粒，分别得敲除GFP基因Crispr/Cas9腺病毒载体，并分别命名为pAdTrack-pSpCas9-T1、pAdTrack-pSpCas9-T2和pAdTrack-pSpCas9-T3。
3、pAd-pSpCas9-T1、pAd-pSpCas9-T2和pAd-pSpCas9-T3的构建
(1)E.coli BJ5183感受态制备：将-80℃冻存的E.coli BJ5183菌株(简称BJ5183)，划线于含链霉素(30～50μg/mL)的LB平板，37℃倒置培养12～16h，然后挑取单克隆摇菌培养，用CaCl2法制作为感受态细胞。
(2)pAdEasy-1质粒转化BJ5183感受态：将pAdEasy-1质粒采用热击法转化步骤(1)制备的E.coli BJ5183感受态，然后涂于含Amp浓度为100μg/mL的LB平板，挑取单克隆于含Amp浓度为100μg/mL的液体LB培养基中，待菌液浑浊后，再用CaCl2法制备含pAdEasy-1质粒的BJ5183感受态。
(3)将pAdTrack-pSpCas9-T1，pAdTrack-pSpCas9-T2和pAdTrack-pSpCas9-T3分别用PmeⅠ酶切线性化，然后用CIAP碱性磷酸酶去磷酸化PmeⅠ单酶切的质粒，防止自我环化。
(4)将步骤(3)去磷酸化后的线性质粒分别采用热击法转化含pAdEasy-1质粒的BJ5183感受态，线性质粒转入含pAdEasy-1质粒的BJ5183感受态后与质粒pAdEasy-1进行重组，分别得pAd-pSpCas9-T1、pAd-pSpCas9-T2和pAd-pSpCas9-T3重组质粒。
同时将pAdTrack-pSpCas9用PmeⅠ酶切线性化，再用CIAP碱性磷酸酶去磷酸化，然后转化含pAdEasy-1质粒的BJ5183感受态，得空载体pAd-pSpCas9(BB)质粒。
4、腺病毒包装
(1)无内毒素质粒DNA的制备
A、分别取提取的pAd-pSpCas9-T1、pAd-pSpCas9-T2、pAd-pSpCas9-T3和pAd-pSpCas9(BB)重组质粒1μL加入100μL DH5α感受态细胞中吹匀，放置冰中静置20min，再放入42℃水浴90s，迅速置于冰浴中3min，加入500μL LB液体培养基，放置摇床180rpm37℃1h，取菌液100μL均匀涂布于Amp浓度为100μg/mL的LB固体培养基37℃培养过夜。
B、取单菌落于3mL Amp浓度为100μg/mL的LB液体培养基中，250rpm、37℃振荡培养8小时；从中取300μL菌液接种于300mL Amp浓度为100μg/mL的LB液体培养基中，并于250rpm、37℃振荡培养12～16小时；
C、收集菌液，然后在4℃、4000rpm条件下离心15min，弃上清，收集菌体，然后按照QIAGEN EndoFree Plasmid Maxi Kit试剂盒说明书操作步骤提取质粒，得无内毒素的pAd-pSpCas9-T1、pAd-pSpCas9-T2、pAd-pSpCas9-T3和pAd-pSpCas9(BB)质粒。
(2)细胞株HEK293的复苏与培养
取液氮冻存的HEK293细胞，迅速放于37℃水浴中解冻，期间不断晃动使离心管内溶液尽量受热均匀；解冻后迅速加入7mL体积分数为10％胎牛血清的DMEM培养液中(DMEM培养液需要提前预热至37℃)，枪头轻轻吹打至无细胞团存在，然后在1300rpm条件下离心6min，弃去上清；再向离心管中加2mL提前预热至37℃体积分数为10％胎牛血清的DMEM培养液，吹打细胞使其悬浮，按照5×104个细胞将细胞接种于培养皿，并于37℃、含5％的CO2培养箱中培养24h，然后换液，以后每隔2天换液至细胞密度达90％时传代。
(3)pAd-pSpCas9-T1、pAd-pSpCas9-T2和pAd-pSpCas9-T3的包装
转染前一天，按照每孔4×105个HEK293细胞接种到6孔板，培养24h后细胞约有70％融合，转染前4h，用无双抗PBS清洗2次细胞，将含血清的培养液换为无双抗无血清的优化培养液Opti-MEMI(2mL/孔)；LipofectamineTM2000(购自Invitrogen公司)作为转染试剂将pAd-pSpCas9-T1转染至HEK293细胞，具体步骤依照说明书进行(所有试剂均为一个孔的试剂用量)：
A.用Opti-MEMI优化培养液将4μg无内毒素的pAd-pSpCas9-T1质粒稀释至250μL，室温孵育；
B.同样用Opti-MEMI优化培养液将10μL LipofectamineTM2000稀释至250μL，室温孵育5min；前两步操作不得超过25min；
C.将孵育的质粒和LipofectamineTM2000混匀，总体积为500μL，整个过程尽量轻柔，混合溶液室温孵育20min；
D.将500μL混合液加到培养板的一个孔中，轻轻晃动培养板混匀培养液；
E.将培养液在37℃、体积分数为5％CO2的培养箱培养，6h后将培养液换为体积分数为10％胎牛血清的DMEM培养液；其中6孔板的4个孔按上述步骤进行包装，另外两个孔一个为脂质体对照，一个为空白对照。
F.转染24h观察细胞病变(Cytopathic effect，CPE)情况，转染8～10d后形成一般会有蚀斑出现，此时可将细胞刮下，将细胞收集在冻存管中，于-196℃(液氮中)和37℃反复冻融5次，12000rpm离心5min，收集的上清液即为包装后的病毒液，命名为Ad-pSpCas9-T1，该病毒液能够直接用于后续细胞感染或于-80℃保存。
按照与上述相同的方法包装pAd-pSpCas9-T2质粒、pAd-pSpCas9-T3质粒和空载体质粒pAd-pSpCas9(BB)，分别得病毒液Ad-pSpCas9-T2、Ad-pSpCas9-T3和Ad-pSpCas9(BB)。
(4)病毒滴度测定
按每孔2×104个细胞的密度接种HEK293于96孔板中，采用TCID50法和Karbers法测定并计算Ad-pSpCas9-T1的病毒滴度。将以倍比(10-1～10-10)稀释的病毒液感染细胞，每孔加100μL病毒稀释液，培养9～10d统计细胞的CEP病变。依照Karbers公式即：T＝10×101+d(s–0.5)TCID50/mL计算病毒得滴度；其中s是阳性比率之和，也就是所用个稀释度之和。d＝log10稀释度＝1(这是对于10倍的稀释度而言)；并且根据公式：T＝1×10X TCID50/mL＝1×10X–0.7PFU/mL，可将病毒滴度换为PFU/mL单位。
经计算病毒液的纯度和滴度如下：
病毒液Ad-pSpCas9-T1，纯度A260/A280＝1.47，滴度2.5×1011；
病毒液Ad-pSpCas9-T2，纯度A260/A280＝1.51，滴度1.7×1011；
病毒液Ad-pSpCas9-T3，纯度A260/A280＝1.48，滴度3.1×1011；
病毒液Ad-pSpCas9(BB)，纯度A260/A280＝1.50，滴度1.6×1011。
5、病毒感染转GFP小鼠细胞
取转GFP小鼠(转GFP小鼠的制备按Okabe M，Ikawa M，Kominami K，Nakanishi T，Nishimune Y.“Green mice”as asource of ubiquitous green cells.FEBS Lett1997；407：313-9报道的方法制备)尾尖建立成纤维细胞系，然后铺板：将对数生长期的细胞消化重悬后，按1×105/L密度接种于12孔板，生长过夜至70～80％铺满12孔板，然后吸出培养液，换新鲜的培养液，分别加入Ad-pSpCas9-T1、Ad-pSpCas9-T2和Ad-pSpCas9-T3的病毒液，感染复数为400，同时以PBS和Ad-pSpCas9(BB)病毒液作为对照，混合均匀后放入孵箱中培养，24小时左右换液，48小时后收集细胞，提取细胞DNA。PCR扩增GFP基因靶点基因序列，PCR扩增上游引物为Cas9-GFP-F：5’-gtgagcaagggcgaggag-3’(SEQ ID NO.10)；下游引物为Cas9-GFP-R：5’-tggtagtggtcggcgagc-3’(SEQ ID NO.11)，PCR产物用T7Endonuclease I酶切，核酸凝胶电泳检测，然后使用BIO-RAD成像系统(ChemiDocXRS)软件分析统计靶点非同源重组修复(NHEJ)效率，结果如图1所示。由图1可知，感染腺病毒Ad-pSpCas9-T2病毒液的细胞GFP基因靶位点NHEJ效率最高为48％，筛选出该病毒液用于转GFP小鼠尾静脉注射。
6、尾静脉注射转GFP基因小鼠
将9只转GFP基因小鼠，随机分为3组，每组3只(1组编号为1、2、3，2组编号为4、5、6，3组编号为7、8、9)，第一组尾静脉注射Ad-pSpCas9-T2病毒液(病毒剂量1×1011PFU/kg)注射，第二组尾静脉注射等剂量的腺病毒Ad-pSpCas9(BB)，第三组注射等量的生理盐水，每7天注射一次病毒，连续注射3次，共计饲养21天。取转GFP小鼠的心、肝、脾、肺、肾组织，并建立细胞系，并用流式细胞仪分析小鼠各组织的细胞，并分选出绿色荧光阴性细胞和阳性细胞，结果如图2所示。结果显示，注射Ad-pSpCas9-T2病毒液各组织均可以分选出不同比例的阴性细胞(图2A)，且注射Ad-pSpCas9-T2病毒液的小鼠主要器官(心、肝、脾、肺、肾)，基因组中GFP基因的敲除效率可以达到30.6％-45.8％；而注射Ad-pSpCas9(BB)病毒液和生理盐水的两组细胞均带有绿色荧光，未分选出绿色荧光阴性细胞(图2B和图2C)。上述结果表明，人工整合敲除GFP基因CRISPR/Cas9元件的腺病毒载体，Ad-pSpCas9-T2病毒液在受体(转GFP小鼠)动物模型，可以有效的靶向修饰靶基因GFP，证实了腺病毒介导CRISPR/Cas9系统能够在活体动物中进行基因靶向的修饰。
将感染Ad-pSpCas9-T2病毒液的转GFP小鼠各组织分选出的绿色荧光阴性细胞和阳性细胞提取DNA，然后PCR扩增GFP基因靶位点序列，扩增片段克隆至pMD18-T载体中，将重组载体送至上海生工生物工程有限公司测序，结果如图3所示。结果显示，绿色荧光阳性细胞中GFP基因序列有部分发生突变，绿色荧光阴性细胞的GFP基因序列均有突变。
实施例2、腺病毒介导CRISPR/Cas9靶向敲除转c-myc基因小鼠
1、构建腺病毒表达载体
利用在线工具ZiFiT Targeter version4.0，在c-myc基因第二外显子(GenBank：AH005318.1)上设计3个CRISPR/Cas9靶点，并设计对应的寡核苷酸，具体为：
第一个靶点为T4，核苷酸序列为5’-tcgctacgtccttctcccca-3’(SEQ ID NO.12)；其设计的寡核苷酸对为5’-caccgtcgctacgtccttctcccca-3’(SEQ ID NO.13)；5’-aaactggggagaaggacgtagcgac-3’(SEQ ID NO.14)；
第二个靶点为T5，核苷酸序列为5’-gcagccgcccgcgcccagtg-3’(SEQ ID NO.15)；其设计的寡核苷酸对为：5’-caccgcagccgcccgcgcccagtgg-3’(SEQ ID NO.16)；5’-aaaccactgggcgcgggcggctgcg-3’(SEQ ID NO.17)；
第三个靶点为T6，核苷酸序列为5’-cagatgatgaccgagttact-3’(SEQ ID NO.18)；其设计的寡核苷酸对为：5’-caccgcagatgatgaccgagttact-3’(SEQ ID NO.19)；5’-aaacagtaactcggtcatcatctgc-3’(SEQ ID NO.20)。
然后按照实施例1的方法分别获得质粒pAdTrack-pSpCas9-T4、pAdTrack-pSpCas9-T5和pAdTrack-pSpCas9-T6。并按照实施例1的方法分别制备Ad-pSpCas9-T4、Ad-pSpCas9-T5和Ad-pSpCas9-T6病毒液，然后测定病毒液滴度，其测定结果如下：
Ad-pSpCas9-T4病毒液，纯度为A260/A280＝1.47，滴度为2.5×1011；
Ad-pSpCas9-T5病毒液，纯度为A260/A280＝1.51，滴度为1.7×1011；
Ad-pSpCas9-T6病毒液，纯度为A260/A280＝1.48，滴度为3.1×1011。
2、病毒液感染受体小鼠
取转c-myc基因小鼠尾尖建立成纤维细胞系(转c-myc基因小鼠的制备按Harris A W，Harris A W,Pinkert C A,Crawford M,et al.The E mu-myc transgenic mouse.A model forhigh-incidence spontaneous lymphoma and leukemia of early B cells[J].The Journal ofexperimental medicine,1988,167(2):353-371.报道的方法制备)，然后铺板，将对数生长期的细胞消化重悬后，按1×105/L密度接种于12孔板，生长过夜至70～80％铺满12孔板，然后吸出培养液，换新鲜的培养液，按照感染复数400particle/cell将细胞分为5组，分别感染病毒液Ad-pSpCas9-T4、Ad-pSpCas9-T5、Ad-pSpCas9-T6、Ad-pSpCas9(BB)和PBS，混合均匀后即可放入孵箱培养24小时换液，72小时后消化细胞，提取细胞DNA。
根据c-myc基因的靶点设计检测引物，具体为，上游引物为M1-F：5’-gagcgagctgcagccgcccgcg-3’(SEQ ID NO.21)，下游引物为M1-R5’-gcccgcgggcggggctcagg-3’(SEQ ID NO.22)。然后以提取的细胞DNA为模板，分别以SEQ ID NO.21和SEQ ID NO.22所示序列为引物进行扩增。将扩增得到的PCR产物用T7核酸内切酶(T7E1)进行酶切，酶切后在80-120V条件下，用琼脂糖凝胶电泳40min检测，结果如图4所示。结果表明，病毒液Ad-pSpCas9-T4对靶点造成的突变的效率最高。
将病毒液Ad-pSpCas9-T4感染的细胞提取DNA，利用c-myc基因的上游和下游引物扩增c-myc基因，扩增产物克隆至pMD18-T载体中，送至上海生工生物工程有限公司测序，结果如图5所示。结果表明病毒液Ad-pSpCas9-T4感染细胞后能够在靶点区域形成突变。
3、转c-myc基因小鼠尾静脉注射腺病毒
取30只转c-myc基因的小鼠(10周龄)，随机分为3组，每组10只，第一组注射病毒液Ad-pSpCas9-T4，第二组和第三组小鼠分别注射病毒液Ad-pSpCas9(BB)和PBS作为对照，每周注射一次，在SPF级动物饲养房中饲养小鼠30周，观察小鼠存活率并记录结果，结果如图6所示。结果显示，尾静脉注射PBS组的小鼠患肿瘤死亡9只，尾静脉注射Ad-pSpCas9(BB)病毒液的小鼠因患肿瘤全部死亡，而尾静脉注射Ad-pSpCas9-T4病毒液小鼠因患肿瘤死亡5只，且死亡时间较。说明注射腺病毒Ad-pSpCas9-T4敲除小鼠c-myc基因，从而能够抑制小鼠肿瘤的发病，延长小鼠的存活时间。
4、转c-myc基因小鼠基因靶位点测序
将3个组死亡小鼠和存活小鼠解剖，取心、肝、脾、肺和肾组织，提取各个组织的DNA为模板，以SEQ ID NO.21和SEQ ID NO.22的序列为引物进行PCR扩增c-myc基因，将PCR产物回收并克隆至PMD18-T载体中，转化后，每只小鼠各组织挑取20个单菌落，送上海生工测序，然后根据测序结果统计非同源重组修改(NHEJ)，结果如表1所示。
表1、死亡小鼠和存活小鼠各组织的非同源重组修改统计结果
由表1可知，实验组的小鼠各个组织器官中的c-myc基因均发生了不同程度的非同源重组修改(NHEJ)。表明腺病毒介导CRISPR/Cas9系统可以成功对转c-myc基因小鼠基因中c-myc基因进行靶向敲除，从而达到抑制小鼠肿瘤发生的目的。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
Claims (8)
1.含有CRISPR/Cas9系统的靶向敲除载体，其特征在于：所述靶向敲除载体由pX330-U6-Chimeric_BB-CBh-hSpCas9质粒用EcoRI和SacII酶切、补平后连接经BstXI酶切、补平的pAdTrack-CMV质粒，然后用BbsI线性化后连入目的基因的特异靶序列，再用PmeⅠ线性化和CIAP碱性磷酸酶去磷酸化后与pAdEasy-1质粒重组而得。
2.根据权利要求1所述含有CRISPR/Cas9系统的靶向敲除载体，其特征在于：所述目的基因为GFP或c-myc基因。
3.根据权利要求2所述含有CRISPR/Cas9系统的靶向敲除载体，其特征在于：目的基因为GFP的特异靶序列如SEQ ID NO.1、SEQ ID NO.4或SEQ ID NO.7所示。
4.根据权利要求2所述含有CRISPR/Cas9系统的靶向敲除载体，其特征在于：目的基因为c-myc基因的特异靶序列如SEQ ID NO.12、SEQ ID NO.15或SEQ ID NO.18所示。
5.根据权利要求4所述含有CRISPR/Cas9系统的靶向敲除载体，其特征在于：所述特异靶序列如SEQ ID NO.12所示。
6.含有权利要求1-5任一项所述靶向敲除载体的腺病毒。
7.权利要求1-5任一项所述靶向敲除载体在制备突变特异靶序列的试剂中的应用。
8.权利要求5所述靶向敲除载体在制备抑制肿瘤的试剂中的应用。
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