CN104762321A - 基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法及其crRNA原件 - Google Patents
基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法及其crRNA原件 Download PDFInfo
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Abstract
基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法及其crRNA原件，涉及一种敲除载体构建方法及其crRNA原件。是要解决现有方法抑制KHVD不足的问题。方法：一、合成crRNA-TK和crRNA-DP引物，沸水处理，退火，得到两个DNA双链退火产物；二、制备pX330-puro载体；三、对pX330-puro载体进行酶切，进行去磷反应，回收线性化的载体；四、对线性化载体与两个退火产物连接，得连接产物；五、将连接产物转入感受态细胞内，提取重组质粒DNA，命名为pX330-puro TK或pX330-puro DP重组载体。本发明基于定向敲除KHV关键基因，靶向精确性高，胞内病毒抑制效果明显。
Description
技术领域
本发明涉及一种敲除载体构建方法及其crRNA原件。
背景技术
鲤鱼疱疹病(Kai herpesvirus disease,KHVD)是由是鲤鱼疱诊病毒(KHV或CyHV-3)感染引起的鲤鱼及锦鲤的一种急性传染疾病，该病的爆发给我国鱼类养殖业造成了严重的经济损失。该病毒为囊膜包被病毒，直径为170-230nm，含有20面体对称核衣壳，属于疱疹科病毒，其遗传物质属于双链DNA。目前针对疫情爆发的补救手段主要仍采用隔离与大量扑杀，尚无有效救治手段。研究证实，实验条件下鲤鱼脑细胞系CCB和鳍条细胞系KF-1是KHV的敏感细胞。
CRISPR：成簇的、规律间隔的短回文重复序列，是原核生物抵抗外来基因片段—噬菌体、质粒等的免疫防御系统。属于III型CRISPR的CRISPR/Cas9系统由三个必要的部分组成：包括crRNA、tracrRNA及Cas9核酸内切酶。目前常见的px330载体中，是将crRNA与tracrRNA进行连接组成含茎环结构的sgRNA。crRNA的5’端含20bp的特异序列，可以识别特定DNA的互补序列。在发挥识别作用过程中，与靶基因前间区3’邻近的3个(NGG)碱基称为PAM序列。在细胞内转染px-330后，含有crRNA的gRNA将Cas9核酸酶带到基因组上的具体靶点，从而对特定基因位点进行切割导致突变，从而实现基因敲除。pX330-U6-Chimenic_BB-CBh-hSpCas9(pX330-puro）转基因供体质粒关键原件示意图如图2所示，关键元件含有组成gRNA的crRNA片段和tracerRNA片段，Cas9蛋白及Puro抗性基因完整表达盒。
目前，CRISPR系统作为高效DNA编辑工具已被大量应用，有报道称该系统可以在细胞水平通过在病毒基因组DNA中造成特异性的双链断裂从而阻抑相应病毒在胞内的复制如属于cccDNA病毒的HBV和感染过程中存在pre-DNA形式的EBV、HIV，不过尚未有采用该策略成功抑制鱼类DNA病毒的报道。
发明内容
本发明是要解决现有方法抑制KHVD不足的问题，提供一种基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法及其crRNA原件。
本发明基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法，按以下步骤进行：
一、依据KHV基因组TK(GenBank:AB375385.1)和DP(GenBank:AY939862.1)基因设计crRNAs，通过http://crispr.mit.edu/设计优化并分别合成crRNA-TK引物和crRNA-DP引物，于沸水处理15min，而后自然降温过夜退火，分别得到两个带有BbsI酶切粘性末端的DNA双链退火产物；
二、为了便于筛选克隆化细胞系，本发明前期工作对能够携带CRISPR/Cas9的pX330载体(pX330-U6-Chimenic_BB-CBh-hSpCas9；http://www.addgene.org/42230)进行了改造，成功制备了含有puromycin抗性基因的pX330-puro载体，如图1所示；
三、对pX330-puro载体采用BbsI进行单酶切，而后采用SAP磷酸酶进行去磷反应，回收线性化的pX330-puro载体；
四、采用T4连接酶，对步骤三回收得到的线性化的pX330-puro载体与步骤一得到的两个退火产物分别进行连接，得连接产物；
五、将步骤四的连接产物转入感受态细胞内，培养后，挑取单菌落进行PCR验证，对PCR验证为阳性的单菌落，提取重组质粒DNA，对重组质粒DNA采用BbsI酶进行单酶切验证，对验证为阳性的重组质粒DNA，命名为pX330-puro TK或pX330-puro DP重组载体。
其中步骤一中crRNA-TK引物序列为：
上游引物：5’-CACCGTGCTCTTGCCCGCGAACAT-3’
下游引物：5’-AAACATGTTCGCGGGCAAGAGCAC-3’
crRNA-DP引物序列为：
上游引物：5’-CACCGCCGTGTTCCTCACGTACTCG-3’
下游引物：5’-AAACCGAGTACGTGAGGAACACGGC-3’
基于CRISPR/Cas9系统靶向敲除KHV基因的特异性crRNA原件为crRNA-TK和crRNA-DP，所述crRNA-TK为双链DNA，其正义链如序列表中的SEQIDNO：1所示，反义链如序列表中的SEQIDNO：2所示；正义链5’-GTGCTCTTGCCCGCGAACAT-3’，反义链5’-ATGTTCGCGGGCAAGAGCAC-3’。
所述crRNA-DP为双链DNA，其正义链如序列表中的SEQ ID NO：3所示，反义链如序列表中的SEQ ID NO：4所示；正义链5’-CCGTGTTCCTCACGTACTCG-3’，反义链5’-CGAGTACGTGAGGAACACGG-3’。
在本发明中，我们应用CRISPR/Cas9系统在鲤鱼鲤鱼鳍条KF-1细胞系中进行了KHV增殖抑制试验。为了有效干扰KHV病毒的复制，我们选取了与病毒复制有关的TK和DP 作为CRISPR/Cas9系统的靶标。为了提高检测的灵敏度，我们根据病毒早期转录的ORF-81基因设计合成了taqman水解探针。结果显示：在鲤鱼鳍条细胞系内，CRISPR/Cas9系统可以在有效的抑制KHV的增殖。TK基因靶向切割示意图如图3所示，Cas9核酸内切酶复合物在gRNA指导下(靶向与crRNA反向互补的双链DNA)可定向在DNA识别区的PAM序列(NGG)前5nt位置造成双链断裂。
本发明的有益效果：
本发明由带有CRISPR/Cas9系统的pX330-puro载体，连接针对KHV基因组TK和DP基因的crRNA确保对其目的DNA片段的特异性破坏。本发明与现有技术相比，具有安全、高效特点，其病毒抑制率能达到80％。本发明具有以下优势：1、靶向精确性高，细胞内病毒抑制效果明显；2、构建简便，实验周期短。
附图说明
图1为含有puromycin抗性基因的pX330-puro载体质粒图谱；图2为转基因供体质粒关键原件示意图；图3为TK基因靶向切割示意图；图4为pX330-puro TK和pX330-puro DP细胞克隆PCR验证结果；图5为KHV病毒拷贝Real-time PCR监测标准曲线；图6为病毒拷贝数监测KHV增殖情况；图7为病毒拷贝数监测KHV增殖情况；图8为连续测定细胞中病毒滴度增减情况。
具体实施方式
本发明技术方案不局限于以下所列举具体实施方式，还包括各具体实施方式间的任意组合。
具体实施方式一：本实施方式基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法，按以下步骤进行：
一、依据KHV基因组TK(GenBank:AB375385.1)和DP(GenBank:AY939862.1)基因设计crRNAs，通过http://crispr.mit.edu/设计优化并分别合成crRNA-TK引物和crRNA-DP引物，于沸水处理15min，而后自然降温过夜退火，分别得到两个带有BbsI酶切粘性末端的DNA双链退火产物；
二、为了便于筛选克隆化细胞系，本发明前期工作对能够携带CRISPR/Cas9的pX330载体(pX330-U6-Chimenic_BB-CBh-hSpCas9；http://www.addgene.org/42230)进行了改造，成功制备了含有puromycin抗性基因的pX330-puro载体，如图1所示，重组后的px330puro载体全长10046bp，如序列表中的SEQ ID NO：21所示；
三、对pX330-puro载体采用BbsI进行单酶切，而后采用SAP磷酸酶进行去磷反应， 回收线性化的pX330-puro载体；
四、采用T4连接酶，对步骤三回收得到的线性化的pX330-puro载体与步骤一得到的两个退火产物分别进行连接，得连接产物；
五、将步骤四的连接产物转入感受态细胞内，培养后，挑取单菌落进行PCR验证，对PCR验证为阳性的单菌落，提取重组质粒DNA，对重组质粒DNA采用BbsI酶进行单酶切验证，对验证为阳性的重组质粒DNA，命名为pX330-puro TK或pX330-puro DP重组载体。
其中步骤一中crRNA-TK引物序列为：
上游引物：5’-CACCGTGCTCTTGCCCGCGAACAT-3’
下游引物：5’-AAACATGTTCGCGGGCAAGAGCAC-3’
crRNA-DP引物序列为：
上游引物：5’-CACCGCCGTGTTCCTCACGTACTCG-3’
下游引物：5’-AAACCGAGTACGTGAGGAACACGGC-3’
步骤三中单酶切反应体系如下：
| 成分 | 用量 |
| 200ng/μLpX330-puro载体 | 5μL |
| Buffer | 3μL |
| BbsI | 2μL |
| ddH2O | 20μL |
酶切反应条件：37℃反应3h。
步骤三中去磷反应条件：线性化的pX330-puro载体30μL，SAP1μL，Buffer5μL，ddH2O14μL，反应30min后，60℃终止SAP作用15min。
步骤四中连接体系如下：
| 成分 | 用量 |
| 线性化的pX330-puro载体 | 0.5μL |
| 退火产物 | 7.5μL |
| T4DNA连接酶 | 1μL |
| Ligase Buffer | 1μL |
连接反应条件：17℃反应12h。
步骤五中pX330-puro TK阳性质粒鉴定引物序列为：
上游引物：5’-CACCGTAAACTGACAGGTCGTGCAT-3’
下游引物：5’-AAACATGTTCGCGGGCAAGAGCAC-3’；
pX330-puro DP阳性质粒鉴定引物序列为：
上游引物：5’-CACCGTAAACTGACAGGTCGTGCAT-3’
下游引物：5’-AAACCGAGTACGTGAGGAACACGGC-5’；
其中各阳性质粒上游检测引物均采用human U6启动子序列(5’-CACCGTAAACTGACAGGTCGTGCAT-3’)，下游引物采用合成各crRNA退火产物的下游引物。检测PCR反应条件如下：94℃预变性10min；94℃变性30s，55℃退火30s，72℃延伸30s，30个循环；循环后72℃延伸10min，于4℃保存。
通过以下实验验证本发明的效果：
实验1：瞬时转染pX330-puro TK和pX330-puro DP抑制KHV的增殖
A、计数KF-1细胞，用含有10％FBS、谷氨酰胺、双抗(100U/ml penicillin；100μg/ml streptomycin)的DMEM营养液以1.0×104/孔传代于24孔培养板，25℃、5％CO2培养24h，使贴壁细胞汇合度达到75％，弃去培养液，用无血清无抗生素DMEM洗涤单层细胞3次，加入450μL无血清无抗生素DMEM备用。
B、接种将含有4.5×102TCID50KHV的DMEM加入每孔，25℃感作2小时后，将培养液换为含有3％FBS，谷氨酰胺，双抗(100U/ml penicillin；100ug/ml streptomycin)的DMEM，培养条件为25℃，5％CO2。
C、染毒后0.5天(0.5dpi)，用LTX regent(Invitrogen)将制备好的2μg pX330-puro TK和pX330-puro DP混合质粒转染鳍条细胞系。采用荧光定量Real-time PCR和病毒的半数细胞感染量测定KHV在转染细胞中的增殖。
基于taqman水解探针检测原理的针对KHV病毒ORF81基因的Real-time PCR检测引物如下：
5’-AGAGGTCTATGCGCGACTAT-3’
5’-FAM-AGACACTGAGAGCGTCATCGGTCA-BHQ1-3’
5’-CACATCTTGCCGGTGTACTT-3’
实验2：稳定表达pX330-puro TK和pX330-puro DP的KF-1细胞抑制KHV增殖
A、用ApaLI核酸内切酶线性化pX330-puro TK和pX330-puro DP载体，冷乙醇沉淀回收备用。
B、计数KF-1细胞，用含10％FBS、无抗生素的DMEM培养液以2×105/孔传代于6 孔板，25℃5％CO2培养48h，使细胞汇合率大于80％，用不含Ca2+，Mg2+离子的PBS洗涤细胞2次，而后每孔加入1.5mL无血清optiMEM。
C、采用LTX regent(Invitrogen)将制备好的线性化载体每孔2μg转染KF-1鳍条细胞系。
D、转染一天后，用含有500ng/ml的puromycin(Sigma aldrich)进行筛选，每2-3天更换培养液。2周后采用克隆环挑取克隆，并进行PCR鉴定。鉴定引物如下：
TK克隆：上游引物：5’-GATTCCTTCATATTTGCATATAC-3’
下游引物：5’-ATGTTCGCGGGCAAGAGCAC-3’
DP克隆：上游引物：5’-GATTCCTTCATATTTGCATATAC-3’
下游引物：5’-CGAGTACGTGAGGAACACGG-3’
检测PCR反应条件如下：94℃预变性10min；94℃变性30s，55℃退火30s，72℃延伸30s，30个循环；循环后72℃延伸10min，于4℃保存。而后在含有EB的1％琼脂糖凝胶进行电泳，检测结果反映克隆化细胞均为阳性，结果见图4。
E、计数克隆化KF-1细胞，均匀铺于96孔板。待细胞汇合度达到50％，将含有100TCID50KHV的DMEM加入每孔，25℃感作2小时后，将培养液换为含有3％FBS，谷氨酰胺，双抗(100U/ml penicillin；100μg/ml streptomycin)的DMEM，培养条件为25℃、5％CO2。每3天更换培养液。
F、接毒7天后采用DNA提取试剂盒(天根)分别提取上清及细胞DNA，同时使用前述taqman水解探针Real-time PCR检测病毒基因组拷贝数。每个反应中加入400nM引物，80nM taqman探针，各样品设三个重复，结果见图5-7；KHV病毒拷贝Real-time PCR监测标准曲线如图5所示，为检测KHV病毒基因组拷贝数，将T-ORF81标准载体连续稀释，应用taqman探针水解法得到相应曲线，图5用于绝对定量病毒拷贝数。病毒拷贝数监测KHV增殖情况如图6，通过Real-time PCR检测方法，以细胞内DNA为模板，7dpi的病毒含量为对照细胞中的1/3，且差异明显，表明导入细胞的瞬转载体有效地抑制了病毒在感染细胞中的增殖。病毒拷贝数监测KHV增殖情况如图7，通过Real-time PCR检测方法，分别采用细胞上清和细胞内DNA为模板，7dpi胞内外的病毒拷贝数都明显的被稳定表达的Cas9和特异gRNA明显抑制。
另外按照Reed–Muench法统计病毒滴度以测定检查KHV在转染细胞及对照细胞中的增殖，分析抗毒原件对KHV感染增殖过程的抑制作用。结果见图8和表1，图8中-○-表示对照，-■-表示TK，-▲-表示DP，-▼-表示TK&DP。图8为连续测定 细胞中病毒滴度增减情况，通过连续统计7-11dpi细胞上清病毒滴度，结果表明：和对照相比，DP和TK&DP克隆细胞上清在10dpi已经不能产生致细胞病变效应，TK克隆上清的病毒亦维持在低水平。
表1测定细胞中病毒的半数细胞感染量TCID50增减情况
| TCID50(8dpi) | TCID50(9dpi) | |
| 对照 | 103.86 | 105.18 |
| CRISPR/Cas9 | 102.52 | 103.44 |
| 和对照比值 | 1/22 | 1/55 |
对照细胞感染KHV后，病毒能有效地在细胞中增殖，显示出较高的感染滴度。
Claims (5)
1.基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法，其特征在于该方法按以下步骤进行：
一、依据KHV基因组TK和DP基因设计crRNAs，分别合成crRNA-TK引物和crRNA-DP引物，于沸水处理15min，而后自然降温过夜退火，分别得到两个带有BbsI酶切粘性末端的DNA双链退火产物；
二、对能够携带CRISPR/Cas9的pX330载体进行了改造，成功制备了含有puromycin抗性基因的pX330-puro载体；
三、对pX330-puro载体采用BbsI进行单酶切，而后采用SAP磷酸酶进行去磷反应，回收线性化的pX330-puro载体；
四、采用T4连接酶，对步骤三回收得到的线性化的pX330-puro载体与步骤一得到的两个退火产物分别进行连接，得连接产物；
五、将步骤四的连接产物转入感受态细胞内，培养后，挑取单菌落进行PCR验证，对PCR验证为阳性的单菌落，提取重组质粒DNA，对重组质粒DNA采用BbsI酶进行单酶切验证，对验证为阳性的重组质粒DNA，命名为pX330-puro TK或pX330-puro DP重组载体。
2.根据权利要求1所述的基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法，其特征在于步骤三中单酶切反应体系如下：
酶切反应条件：37℃反应3h。
3.根据权利要求1所述的基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法，其特征在于步骤三中去磷反应条件：线性化的pX330-puro载体30μL，SAP 1μL，Buffer 5μL，ddH2O 14μL，反应30min后，60℃终止SAP作用15min。
4.根据权利要求1所述的基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法，其特征在于步骤四中连接体系如下：
连接反应条件：17℃反应12h。
5.基于CRISPR/Cas9系统靶向敲除KHV基因的crRNA原件，其特征在于crRNA原件为crRNA-TK和crRNA-DP，所述crRNA-TK为双链DNA，其正义链如序列表中的SEQID NO：1所示，反义链如序列表中的SEQ ID NO：2所示；所述crRNA-DP为双链DNA，其正义链如序列表中的SEQ ID NO：3所示，反义链如序列表中的SEQ ID NO：4所示。
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