CN107519492A - 使用CRISPR技术敲除miR‑3187‑3p在冠状动脉粥样硬化性心脏病中的应用 - Google Patents
使用CRISPR技术敲除miR‑3187‑3p在冠状动脉粥样硬化性心脏病中的应用 Download PDFInfo
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Abstract
本发明提供一种利用CRISPR‑Cas9特异敲除miR‑3187‑3p基因的方法，所述方法为：（1)构建Cas9表达载体；(2构建sgRNA表达载体;(3)将Cas9、sgRNA‑混合成混合液，注射哺乳类动物受精卵，实现将miR‑3187‑3p基因定点敲除的目的。
Description
技术领域
本发明涉及CRISPR技术在冠状动脉粥样硬化性心脏病中的应用，属于医药生物技术领域。
背景技术
冠状动脉粥样硬化性心脏病是冠状动脉血管发生动脉粥样硬化病变而引起血管腔狭窄或阻塞，造成心肌缺血、缺氧或坏死而导致的心脏病，常常被称为“冠心病”。但是冠心病的范围可能更广泛，还包括炎症、栓塞等导致管腔狭窄或闭塞。世界卫生组织将冠心病分为5大类：无症状心肌缺血(隐匿性冠心病)、心绞痛、心肌梗死、缺血性心力衰竭(缺血性心脏病)和猝死5种临床类型。临床中常常分为稳定性冠心病和急性冠状动脉综合征。
在临床表现上主要体现在典型胸痛。因体力活动、情绪激动等诱发，突感心前区疼痛，多为发作性绞痛或压榨痛，也可为憋闷感。疼痛从胸骨后或心前区开始，向上放射至左肩、臂，甚至小指和无名指，休息或含服硝酸甘油可缓解。胸痛放散的部位也可涉及颈部、下颌、牙齿、腹部等。胸痛也可出现在安静状态下或夜间，由冠脉痉挛所致，也称变异型心绞痛。如胸痛性质发生变化，如新近出现的进行性胸痛，痛阈逐步下降，以至稍事体力活动或情绪激动甚至休息或熟睡时亦可发作。疼痛逐渐加剧、变频，持续时间延长，祛除诱因或含服硝酸甘油不能缓解，此时往往怀疑不稳定心绞痛。
目前，治疗冠状动脉粥样硬化性心脏病主要使用抗心绞痛药物，比如硝酸酯类，肾上腺素β受体阻断药，钙通道阻滞剂等。CN105816722A中公开了一种治疗冠状动脉粥样硬化性心脏病的中药制剂，其原料药包括川芎、桃仁、红花、赤芍、当归、土鳖虫、九香虫、川牛膝、红曲、生山楂、雄黑豆、穿山甲、延胡索、醋香附、银杏叶、降香、茯神、远志、乌枣、甘松、琥珀、五爪龙、柴胡、葛根、桔梗、乌贼鱼墨囊。该中药制剂针对病因病机组方用药，具有辨证论治的优势，克服了传统西药无法区分证型进行对证治疗，通过中药的内服，可以在一定程度上替代西药的使用，降低药物依赖性及副作用等。CN 101332246 B中公开了治疗冠心病和心绞痛的中药制剂，主要包括丹参18-22、三七4-6、赤芍17-23、降香13-18、元胡13-18、红花7-11、何首乌18-22、鸡血藤25-35、没药18-22、人参13-17、肉桂13-17、淫羊藿18-22、鹿茸4-6、灵芝11-15、冬虫夏草7-9、川芎12-18、桃仁8-12、乳香13-17、全蝎4-6、蜈蚣4-6、丹皮12-17等组分，根据实验结果来看具有一定的效果。
在现有技术中，CN 103877576 B中公开了以Card3基因敲除小鼠和心脏特异性Card3转基因小鼠为对象，通过阻断小鼠心脏左冠状动脉前降支造成心肌梗死模型进行研究，结果表明与WT小鼠对比，Card3基因敲除小鼠心脏梗死比例、心肌肥厚和纤维化的程度明显被抑制，心功能明显好转；CN 103893743 B中以GDF1基因敲除小鼠和心脏特异性GDF1转基因小鼠为实验对象，通过阻断小鼠心脏左冠状动脉前降支造成心肌梗死模型进行研究，结果表明与MEM-Cre对照小鼠对比，GDF1基因敲除小鼠心脏梗死比例、心肌肥厚和纤维化的程度明显增加，心功能明显恶化；而心脏特异性GDF1转基因小鼠的心脏梗死比例、心肌肥厚和纤维化的程度明显被抑制，心功能明显改善。CN 103898189 B中公开了以SHPS-1基因敲除小鼠和心脏特异性SHPS-1转基因小鼠为实验对象，通过阻断小鼠心脏左冠状动脉前降支(LAD)造成心肌梗死模型，结果表明与WT对照小鼠对比，SHPS-1基因敲除小鼠在MI术后心脏梗死比例、心肌肥厚和纤维化的程度明显被抑制，心功能明显好转；CN 103893763 B中公开了以Vinexin-β基因敲除小鼠和心脏特异性Vinexin-β转基因小鼠为实验对象，通过阻断小鼠心脏左冠状动脉前降支(LAD)造成心肌梗死模型，结果表明与WT对照小鼠对比，Vinexin-β基因敲除小鼠在MI术后心脏梗死比例、心肌肥厚和纤维化的程度明显被抑制，心功能明显好转。通过以上结果可以发现，通过基因干扰可以实现冠状动脉粥样硬化性心脏病的治疗。
目前，规律成族间隔短回文重复系统(clustered regularly interspaced shortpalindromic repeat；CRISPR-associated，CRISPR_Cas9)是一种具有核酸内切酶活性的复合体，识别特定的DNA序列，进行特定位点切割造成双链DNA断裂(Double-strand breaks，DSB)，在没有模板的条件下，发生非同源重组末端连接(Non-homologous end joining，NHEJ)，造成移码突变(frameshift mutation)，导致基因敲除。这一技术由于能快速、简便、高效地靶向基因组任何基因，从而引起了广泛的关注，在2012年开始像爆炸一般流行开来。由于其容易操作、可以同时靶向多个基因，可以高通量制备、造价低等优势，Cas9已经成为一种发展最快的技术。正是由于其优越性，这一技术在Nature推荐的2013十大进展中位列第一。
Cas9靶向切割DNA是通过两种小RNA--crRNA(CRISPR RNA)和tracrRNA(trans-activating crRNA)和靶序列互补识别的原理实现的。现在已经把两种小RNA融合成一条RNA链，简称sgRNA(single guide RNA)。因此，sgRNA能否做到特异性、精确靶向目标基因是CRISPR-Cas9能否特异性敲除目标基因的先决条件，无论是脱靶还是错误靶向，都会影响CRISPR-Cas9对目标基因的特异性敲除。因此，能够设计、制备出精确性和特异性靶向目标基因的sgRNA成为CRISPR-Cas9基因敲除的关键技术。与ZFN和siRNA相比，CRISPR-Cas9具有更快速、简便、高效、多位点、特异性靶向敲除基因的优势。
发明内容
根据第一方面，本发明的目的是针对现有技术中的不足，提供微小核苷酸miR-3187-3p采用CRISPR/Cas9系统敲除的目的。这是首次利用CRISPR/Cas9系统特异性的将micrRNA家族的miR-3187-3p进行敲除。利用这种新的方法可高效的实现miR敲除的目的。
本发明另外一方面，根据申请人前期通过筛选获得miR-3187-3p与Card3和SHPS-1的正向表达调节关系，提供了miR-3187-3p的敲除后抑制Card3和SHPS-1基因的表达，从而治疗冠状动脉粥样硬化性心脏病。
本发明另外提供一种sgRNA，其序列为根据miR-3187-3p前体序列，通过前期40多个设计的sgRNA靶点中通过初步试验找到的唯一一个具有较好敲除效果的sgRNA，其序列如ttgacgattagtacagcattc(SEQ ID NO：1所示)。
本发明提供一种利用CRISPR-Cas9特异敲除microRNA基因的方法，所述方法为：(1)构建Cas9表达载体；(2构建sgRNA表达载体；(3)将Cas9、sgRNA-混合成混合液，注射哺乳类动物受精卵，实现将microRNA基因定点敲除的目的。
与现有技术相比，本发明的有益效果主要体现在：
1)基因修饰效率较高，降低了传统技术的不可靠性；2)操作技术简单，无需通过复杂的打靶载体构建、ES细胞筛选、嵌合体小鼠选育等一系列步骤；3)通过简单的一步即可实现复杂的心脏病的治疗，具有极大的市场应用前景。
附图说明
图1lentiCRISPR v2载体图。
图2(1)、(2)、(3)分别为miR-3187-3p、Card3和SHPS-1基因敲除前后的mRNA表达量对比图。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
下述实施例中所用材料、试剂等，如无特别说明，均可从商业途径获得。
实施例1 引物设计
1、构建体外Cas9表达载体，命名为pGEM-Cas9,由赢润生物负责合成，其序列为本领域常用的序列。
2、体外转录sgRNA骨架编码序列的载体构建与制备。
针对miR-3187-3p基因设计sgRNA，正向寡核苷酸序列和反向寡核苷酸序列可以互补形成具有粘性末端的双链DNA片段：
F：CACCGttgacgattagtacagcattc
R:CgaatgctgtactaatcgtcaaCAAA。
实施例2、构建mmu-miR-3187-3p基因的sgRNA表达载体
1.合成DNA插入片段
(1)合成上述设计的正向和反向寡核苷酸序列
寡核苷酸序列可以由商业化的公司(上海生工公司)根据提供的序列具体合成。
将对应的正向和反向寡核苷酸序列退火、复性，形成具有粘性末端的双链DNA片段。
反应体系(20μL)如下所示：
正向寡核苷酸(10μM)：1μL
反向寡核苷酸(10μM)：1μL
10×PCR buffer：2μL
ddH2O：16μL
将上述反应体系放入PCR仪，并按以下程序进行反应。
反应程序：
95℃,5min；
80℃,5min；
70℃,5min；
59℃,5min；
50℃,5min；
自然降至室温。
2.构建sgRNA表达载体
(1)利用BsmB I限制性内切酶酶切目标载体lentiCRISPR v2质粒(优宝生物，货号：VT8107)。
按照以下反应体系进行配制：
LentiCRISPR v2质粒：1μg
10×酶切buffer：2μL
BsmB I限制性内切酶：2μL
补充ddH2O至总体积20μL
将酶切反应体系置于37℃反应3h。
(2)电泳分离并纯化载体片段
酶切结束后，将酶切混合物通过琼脂糖凝胶电泳进行分离，选择载体片段(约12kb)进行切割，并通过DNA凝胶回收柱进行回收。
(3)将合成的双链DNA片段与载体主片段进行连接并转化大肠杆菌
将复性得到的双链DNA片段与回收得到的载体片段进行连接反应，按照以下反应体系进行配制：
LentiCRISPR v2载体片段：100ng
双链DNA片段：200ng
T4连接酶：1μL
T4连接反应buffer：1μL
补充ddH2O至总体积10μL
将连接混合物置于25℃反应2h。
反应结束后将连接混合物转化大肠杆菌DH5α菌株：向连接混合物中加入100μL大肠杆菌DH5α感受态细胞，冰上孵育30min；将混合物放入42℃水浴，热激90s后放入冰上冷却；向混合物加入100μL LB培养基，37℃摇床培养20min；将混合物涂Amp LB平板，37℃培养14h。
(4)鉴定正确的转化克隆
从Amp LB平板上挑选若干菌落进行扩大培养，提取质粒进行酶切鉴定。挑选可能正确的克隆进行测序，通过测序，发现插入序列正确。对于正确的lentiCRISPR v2-sgRNA载体克隆进行保种，并且提取相应的质粒。
实施例3、转基因小鼠制备
1)CRISPR/Cas9注射小鼠体系如下：
| pGEM-Cas9 | 50ng/μl |
| lentiCRISPR v2-sgRNA | 10ng/μl |
| 混合总体积 | 20μl |
2)注射
利用Eppendorf2xTransferManNK2显微注射仪吸取2μl步骤1)混合液注射60个受精卵。随后受孕。
小鼠出生5天后，剪取小鼠指甲抽取基因组DNA。PCR鉴定miR-3187-3p以及Card3和SHPS-1基因表达水平，以未进行基因敲除的小鼠30只作为对照。如图2所示，与对照相比，小鼠体内的miR-3187-3p以及Card3和SHPS-1基因表达水平相对于对照，表达水平分别降低到0.4％、15.7％、16.5％。这充分说明，小鼠体内的miR-3187-3p已经被完整的敲除，基因表达已经几乎完全丧失。对应的，Card3和SHPS-1基因表达水平也显著地降低。
将60只小鼠解剖，miR-3187-3p敲除后的小鼠，心脏显著变小，平均体积为未敲除的小鼠的66.8％。这充分说明，通过miR-3187-3p的敲除可以用于做理疗冠状动脉粥样硬化性心脏病。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
序列表
<110> 侯冬雪
<120> 使用CRISPR技术敲除miR-3187-3p在冠状动脉粥样硬化性心脏病中的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
ttgacgatta gtacagcatt c 21
<210> 2
<211> 26
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
caccgttgac gattagtaca gcattc 26
<210> 3
<211> 26
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
cgaatgctgt actaatcgtc aacaaa 26
Claims (4)
1.miR-3187-3p在制备下调Card3和SHPS-1基因表达的试剂中的应用；其中所述试剂能够治疗冠状动脉粥样硬化性心脏病。
2.miR-3187-3p的敲除试剂在制备用于治疗冠状动脉粥样硬化性心脏病的药物的用途。
3. 如权利要求2的用途，其中敲除采用CRISPR/CAS技术，其中采用的sgRNA序列如SEQID NO：1所示。
4.一种治疗冠状动脉粥样硬化性心脏病的方法，其特征在于：采用CRISPR/CAS技术敲除miR-3187-3p。
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