CN106701763A - CRISPR/Cas9靶向敲除人乙肝病毒P基因及其特异性gRNA - Google Patents
CRISPR/Cas9靶向敲除人乙肝病毒P基因及其特异性gRNA Download PDFInfo
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Abstract
本发明属于基因工程技术领域，更具体地说，本发明涉及基于CRISPR/Cas9系统的向导RNA（gRNA）序列及其组合在用于特异性靶向敲除乙型肝炎病毒cccDNA P基因的gRNA。本发明根据CRISPR/Cas9的设计原则，设计了30个gRNA，其序列表见SEQ ID NO.1‑30所示，并且将其构建在PX458载体上，筛选出4个最高效率的gRNA。在人肝癌细胞株(HepG2.2.15)中利用这4条gRNA及其组合指导的CRISPR/Cas9系统，可以有效的敲除人乙型肝炎病毒cccDNA P基因。利用本发明制备的特异性靶向乙型肝炎病毒cccDNA的gRNA能够精确靶向乙型肝炎病毒 cccDNA 并且实现基因敲除。该制备方法操作简单、gRNA 靶向性好，CRISPR/Cas9系统的敲除效率高。
Description
技术领域
本发明属于基因工程技术领域，具体涉及CRISPR/Cas9特异性敲除人乙肝病毒P基因的方法以及用于特异性靶向人乙肝病毒P基因的gRNA。
背景技术
乙型肝炎病毒（HepatitisBvirus，HBV）是一种DNA病毒，属于嗜肝DNA病毒科，HBV能够引发慢性肝炎、急性肝炎、肝硬化、肝癌等疾病。HBV是世界范围性的流行性疾病，全球约有3.5～4亿人感染慢性HBV，在我国，HBV的感染率高达60%~70%，有9300万人携带乙肝病毒，其中慢性乙型肝炎患者约2000万例。乙型肝炎治疗的目的是减轻肝脏病变，防止或延缓发展为肝硬化、肝癌，最终目标是彻底清除病毒，达到完全治愈。除一般的保护肝脏和免疫调节治疗外，抗病毒治疗是肝炎治疗的重点也是难点。到目前临床应用的抗HBV药物有两大类: 干扰素及核酸类似物，由于HBV易突变，耐药突变株在不断的出现，使得现有的抗HBV药物难以达到理想的治疗效果。
HBV基因组结构高度浓缩，结构基因与调节基因之间重叠，甚至结构基因序列之间也相互重叠，有不同的变异株，但每个病毒株的L(-)链均含有4个开放读码框，分别是S、C、P、X区。其中P-ORF是HBVDNA序列中最长的ORF，编码的P蛋白是对病毒生活周期起重要作用的多功能酶，参与病毒基因组复制的全过程，每一个结构域在基因组复制过程中都发挥不同的作用。由于P蛋白的重要性，使其成为抗病毒药物的主要靶点。
CRISPR-Cas9系统已经成功应用于植物、细菌、酵母、鱼类及哺乳动物细胞，是目前最高效的基因组编辑系统，具有的快速、简便、高效、多位点、特异性靶向敲除基因的优势，为高效靶向敲除HBV P-ORF，实现乙型肝炎及其相关疾病的治疗提供了一种可能的选择。本发明的目的就是要验证利用CRISPR-Cas9 高效靶向敲除HBV P-ORF，提供相应的技术方案，达到特异性敲除HBV P-ORF的目的。
发明内容
本发明的目的在于通过设计、构建、筛选，最终提供一些基于CRISPR/Cas9系统，同时靶向人乙型肝炎病毒P基因的高效gRNA及其靶位点序列，并用其抑制人乙型肝炎病毒P基因的表达，从而抑制乙型肝炎病毒的增值。
为实现上述目的，本发明以CRISPR/Cas9系统原理及其gRNA的设计原理为基础，软件设计预测，设计出一系列的gRNA，并以PX458为表达载体，构建了gRNA/Cas9表达系统。通过筛选和一系列的分析测试，最终筛选出4个针对基因组靶点有效的gRNA，并在HepG2.2.15细胞模型中加以应用。
本申请的技术方案如下
1、 靶向人乙型肝炎病毒P基因的高效gRNA及其靶点序列的设计及gRNA/Cas9表达系统构建；
2、在HepG2.2.15细胞模型中，分析检测gRNA指导的CRISPR系统对于人乙肝病毒cccDNA靶位点敲除效率，筛选到8条较高效的gRNA；
3、在HepG2.2.15细胞模型中，进一步分析检测gRNA指导的CRISPR系统对于人乙肝病毒增殖标志物外膜蛋白HBsAg和核壳蛋白HBeAg的抑制作用，最终筛选到4条较高效率的gRNA，其对应的核酸序列如序列表SEQ ID NO. 1、SEQ ID NO. 3、SEQ ID NO. 10和SEQ ID NO.11任意一条序列所示。
附图说明
图1 为T7 Endonuclease I 酶切结果图；
图2 HepG2.2.15细胞上清中HBsAg和HBeAg测定结果图。
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 靶向人乙肝病毒P基因的gRNA合成及CRISPR/Cas9系统载体构建
1、靶向人乙肝病毒P基因的gRNA 的选择和设计
在Genebank中找到人乙肝病毒P基因的序列，在人乙肝病毒P基因设计潜在靶位点。
通过在线设计工具（http://crispr.mit.edu/）及gRNA的设计原则，评估人乙肝病毒P基因序列上得分较高的靶位点设计gRNA。
2、靶向人乙肝病毒P基因的gRNA 寡核苷酸序列的合成和真核表达载体的构建
将pSpCas9(BB)-2A-GFP (PX458)质粒（Addgene plasmid ID: 48138，以下简称pSpCas9(BB) ），用BbSI酶切，37℃水浴1h后， 1%的琼脂糖电泳，回收酶切产物（TAKARA胶回收试剂盒）。
酶切体系如下：
将两寡核苷酸退火，形成带有粘性末端的短双链DNA，反应体系如下：
将上述反应体系在200 μLPCR管中混合均匀，然后将PCR管在37℃水浴锅中处理30min，再放入500 ml沸水中，自然冷却至室温。
连接体系：
将带有粘性末端的双链短DNA产物连入酶切后的pSpCas9(BB)线性片段，将连接产物转化大肠杆菌DH5α感受态细胞（Takara Code : D9057A），并涂布于氨苄青霉素浓度为100 μg/mL的LB固体平板上培养过夜，挑取生长良好的单克隆，于15 mL氨苄青霉素浓度为100 μg/mL的LB液体培养基中，37℃振荡培养过夜，提取质粒，命名为PX458-gRNA-P1。
3、无内毒素质粒DNA 的制备
A、取PX458-gRNA-P1质粒1 μL加入100 μL DH5α感受态细胞中吹匀，冰中静置20 min，再放入42℃水浴90s，迅速置于冰浴中3 min，加入500 μL LB液体培养基，放置摇床180 rpm37℃ 1 h，取菌液100 μL均匀涂布于氨苄青霉素浓度为100 μg/mL 的LB固体培养基37℃培养过夜；
B、取单菌落3mL 于氨苄青霉素浓度为100 μg/mL的LB液体培养基中，250 rpm、37℃振荡培养8 h；从中取300 μL菌液接种于300 mL 氨苄青霉素浓度为100 μg/mL的LB液体培养基中，并于250 rpm、37℃振荡培养12~16 h；
C、收集菌液，然后在4℃、4000 rpm条件下离心15 min，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PX458-gRNA-P1质粒。
采用同样的操作方法，构建出PX458-gRNA-P2、PX458-gRNA-P3...... PX458-gRNA-P30载体。
实施例2 转染HEPG2.2.15细胞
复苏人肝癌细胞(HEPG2.2.15细胞，中科院上海细胞库)，将细胞放入加有10%的FBS+DMEM培养瓶中，于37℃、5% CO2的培养箱中培养，转染前一天，传代培养细胞。
将培养HEPG2.2.15 T75瓶中的培养基吸净，加入2 mL 4℃冰箱取出的0.25%胰酶，使其均匀覆盖瓶底，置于37℃培养箱中3~5 min，取出，摇晃可发现细胞于底部脱离，将其全部晃下，加入3 mL 37℃水浴中预热的10％DMEM，用10 mL移液管进行吹打，吹打6~8次，不留死角，瓶口处较难吹打可将移液管对准培口，小力将培养基打出即可覆盖到接近瓶口的细胞。之后，将所有细胞吸出，置于15 mL离心管中，取50 μL混匀后的细胞于1.5 mLeppendorf管中，加入450 μL 10％DMEM，即为10倍稀释，混匀，取10 μL细胞于计数板中计数。传代当天记为第一天，若第二天进行转染，铺900~1000万/T75；若第三天转染，铺350~400万/T75。每瓶T75加10 mL 10％DMEM培养基。转染当天观察细胞密度，80~90％满即可进行转染。
采用脂质体转染法将PX458-gRNA-P1转染HEPG2.2.15细胞。转染体系及试剂使用Lipofectamine™ 2000（invitrogen公司），转染详细步骤参照转染说明书。
转染48 h后，利用胰酶消化转染后贴壁的细胞，离心收集细胞，吸掉废液加入1mLPBS重悬细胞，取500 μL放入原瓶中继续培养，剩余细胞放入1.5 mL离心管，提取DNA（按照DNA提取试剂盒说明书进行）。
以提取的DNA为模板（未转染细胞DNA为对照组），扩增靶点序列。验证靶点序列突变情况，来确定PX458-gRNA-P1载体在细胞内的活性。
PCR反应体系如下：
PCR扩增程序：95℃预变性3 min；95℃变性30 s，58℃退火30 s，72℃延伸1 min，30个循环后72℃延伸5 min，最后4℃保温。
PCR扩增引物序列如序列表SEQ ID NO.31-32所示。
PX458-gRNA-P2、PX458-gRNA-P3...... PX458-gRNA-P30载体采用同样的操作方法进行转染以及相关的检测。
将30个载体的PCR产物分别用T7 Endonuclease I 37℃水浴酶切1h，酶切体系如下：
T7 Endonuclease I 酶切PCR产物，琼脂糖凝胶电泳检测结果。电泳检测显示，1、3、8、9、10、11、13、26载体可以成功切开靶位点，筛选该8个载体用于抑制HEPG2.2.15细胞中乙肝病毒增殖。
实施例3酶联免疫吸附法检测HepG2.2.15细胞上清中HBsAg和HBeAg水平
培养HEPG2.2.15细胞并将细胞铺至6孔板，将实施例2筛选获得的8个高效靶向敲除的乙肝病毒的载体，分别转染铺6孔板的细胞，每个载体做3个重复孔，使用等量PBS替代载体（阳性对照），具体转染方法参照实施例2。
转染后24小时后换液，取细胞上清液，离心去电上清中的细胞体或碎片，取转染72h后的细胞上清，5000 rpm 离心去掉上清中细胞胞体或碎片，按照乙型肝炎病毒表面抗原（HBsAg）和e抗原诊断试剂盒(HBsAg)（酶联免疫法，艾康生物技术有限公司）使用说明书进行操作，分别对上清原液测定HBsAg和HbeAg，培养基设为阴性对照组。使用酶标仪读取各实验样品及梯度稀释标准样品的450nm波长OD值（OD 450）。
按照试剂盒说明书计算得 HBsAg和HBeAg阳性临界OD 450值分别为0.112和0.105，本研究中所测样品的HBsAg和HBeAg均为阳性，高于临界值。
具体结果（见图2），显示载体1、3、10、11转染组对于人乙肝病毒HBsAg和HbeAg的抑制效率最高，载体8、12、13、26载体转染组对于人乙肝病毒HBsAg和HbeAg的抑制效率较弱。因此，我们选用1、3、10、11载体以及该载体靶向的人乙肝病毒p基因的靶位点，作为药物开发应用的靶点。
SEQUENCE LISTING
<110> 重庆高圣生物医药有限责任公司
<120> CRISPR/Cas9 靶向敲除人乙肝病毒P基因及其特异性gRNA
<130> 2016
<160> 30
<170> PatentIn version 3.3
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<212> DNA
<213> 人工序列
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agcacaacat caggattcct 20
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ggcctatatt ttcctgctgg 20
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accccgcctg taacacgagc 20
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aggacccctg ctcgtgttac 20
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tcgtcaatct tctcgaggac 20
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gtgaggcagt agtcggaaca 20
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<212> DNA
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acccctgctc gtgttacagg 20
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gaagattgac gatatgggtg 20
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gtcaatcttc tcgaggactg 20
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Claims (4)
1.在CRISPR/Cas9特异性敲除乙型肝炎病毒cccDNA P基因的gRNA，所述 gRNA 在乙型肝炎病毒cccDNA P基因的靶序列符合 5’- N (20)-NGG3’ 或 者 5’-CCN- N (20)N-3’的序列排列规则，在乙型肝炎病毒cccDNA P基因上的靶序列是唯一的，其特征在于：所述gRNA 在人乙型肝炎病毒P基因的靶向位点位于人乙型肝炎病毒P基因外显子上。
2.根据权利要求 1所述的在CRISPR/Cas9特异性靶向敲除乙型肝炎病毒cccDNA P基因的gRNA，其特征在于：其对应的核酸序列如序列表SEQ ID NO. 1、SEQ ID NO. 3、SEQ IDNO. 10和SEQ ID NO. 11任意一条序列所示。
3.根据权利要求1-2所述的在CRISPR/Cas9特异性靶向敲除乙型肝炎病毒cccDNA P基因的gRNA，其特征在于：该gRNA在乙型肝炎治疗中具有广泛的应用前景。
4.CRISPR-Cas9 特异性敲除乙型肝炎病毒cccDNA P基因的方法，具体涉及如下步骤 ：
（1）如权利要求 1-3 任意一项所述的 gRNA，在其对应DNA 序列的5’末端加上 CACC得到正向寡核苷酸序列，在其互补链的 5’末端加上 AAAC 得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列，然后将合成的序列变性、退火，得到具有 BbsI 粘性末端的双链 DNA 片段；
（2）将步骤（1）中合成的双链 DNA 片段和用 BbsI 酶切过的PX458 载体进行连接，将连接产物转化到大肠杆菌DH5a中，涂布于带有氨苄青霉素抗性的LB平板上，筛选阳性菌落，提取阳性菌落质粒进行分析及测序，确定gRNA 表达载体构建成功，命名为PX458-gRNA-P1；
（3）将步骤（2）构建的gRNA载体转染HepG2.2.15细胞，并用PX458空载体转染HepG2.2.15 细胞作为对照组。
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