CN116790603B - 一种sgRNA、CRISPR/Cas9载体及其构建方法和用途 - Google Patents
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Abstract
本发明属于分子技术领域，具体涉及一种sgRNA、CRISPR/Cas9载体及其构建方法和用途。本发明通过特定的sgRNA构建的CRISPR/Cas9***，对五指山猪耳成纤维细胞的NOTCH1和eNOS进行编辑，造成了片段缺失，导致移码敲除，获得了NOTCH1单等位基因编辑（即NOTCH1+/‑）和eNOS双等位基因编辑（即eNOS‑/‑）的猪耳成纤维细胞，可用于制备“NOTCH1+/‑和eNOS‑/‑”基因编辑猪，作为动脉粥样硬化动物模型用于医学和药物研发。
Description
技术领域
本发明属于分子技术领域，具体涉及一种sgRNA、CRISPR/Cas9载体及其构建方法和用途。
背景技术
NOTCH1是成人动脉内皮细胞中表达的主要Notch受体，有研究表明，内皮NOTCH1表达的减少是血管炎症发作和动脉粥样硬化开始的诱发因素，高脂血症会显著下调内皮中Notch1的表达，促动脉粥样硬化因子（Ox-PAPC、TNF和IL-1β）抑制了人EC中Notch1表达，此外，在无外部刺激时，NOTCH1信号传导的减少促进了单核细胞在体外和体内与ECs的结合，并导致促炎和致动脉粥样硬化分子（IL8、CXCL1、SELE、CHST1和TDAG51）的增多。
内皮一氧化氮合酶(eNOS)催化生成的一氧化氮(NO)是脉管***中的关键信号分子，对维持正常的血管内平衡具有重要作用，它能抑制心血管***中白细胞-内皮粘附、血管平滑肌细胞增殖和低密度脂蛋白氧化以及血小板聚集等，被认为具有心血管保护和抗AS功能。研究表明，eNOS基因敲除的小鼠中血压升高且加速了动脉粥样硬化。在eNOS/载脂蛋白E(ApoE)双基因敲除小鼠中，动脉粥样硬化加剧。在高脂饲料诱导的apoE-KO小鼠中，eNOS缺乏会加剧动脉粥样硬化，并导致冠心病和一系列心血管并发症。
目前已有分别将NOTCH1基因和eNOS基因进行基因编辑诱导动脉粥样硬化疾病的报道，且均是以小鼠为模型构建，至今，尚未见NOTCH1基因和eNOS基因同时被基因编辑的动物模型，更没有相比于啮齿类动物，以能更准确地预测人类疾病的猪作为模型，对NOTCH1和eNOS基因进行基因编辑，实现对动脉粥样硬化研究的报道。
发明内容
为解决上述问题，本发明提供了靶向eNOS和NOTCH1基因的sgRNA，它包括核苷酸序列如SEQ ID NO.3 或SEQ ID NO.10所示的eNOS-sgRNA6，和核苷酸序列如SEQ ID NO .6 或SEQ IDNO.13所示的NOTCH1-sgRNA3。
本发明还提供了一种CRISPR/Cas9载体，它是连接有与eNOS-sgRNA6对应的双链DNA的载体，和连接有与NOTCH1-sgRNA3对应的双链DNA的载体；
所述eNOS-sgRNA6的核苷酸序列如SEQ ID NO.3 或SEQ ID NO.10所示；所述NOTCH1-sgRNA3的核苷酸序列如SEQ ID NO .6 或SEQ ID NO.13所示。
进一步地，所述载体为pX458质粒载体。
本发明连接有与eNOS-sgRNA6对应的DNA序列的载体可以在细胞内表达生成eNOS-sgRNA6；连接有与NOTCH1-sgRNA3对应的DNA序列的载体可以在细胞内表达生成NOTCH1-sgRNA3；
其中，eNOS-sgRNA6是引导Cas9对eNOS基因定点编辑的sgRNA；
NOTCH1-sgRNA3是引导Cas9对NOTCH1基因定点编辑的sgRNA。
本发明还提供了一种前述CRISPR/Cas9载体的构建方法，它包括如下步骤：
取与eNOS-sgRNA6对应的双链DNA，***到pX458质粒载体中；取与NOTCH1-sgRNA3对应的双链DNA，***pX458质粒载体中，即得；
所述eNOS-sgRNA6的核苷酸序列如SEQ ID NO.3或SEQ ID NO.10所示；所述NOTCH1-sgRNA3的核苷酸序列如SEQ ID NO .6或 SEQ ID NO.13所示。
本发明还提供了一种前述CRISPR/Cas9载体在制备NOTCH1和eNOS基因敲除的细胞株中的用途。
进一步地，所述细胞株是NOTCH1单等位基因敲除和eNOS双等位基因敲除的细胞株。
进一步地，所述细胞株包括五指山猪耳成纤维细胞。
本发明最后提供了一种NOTCH1和eNOS基因敲除的细胞株的构建方法，它包括如下步骤：
取前述CRISPR/Cas9载体转染五指山猪耳成纤维细胞，富集带绿色荧光细胞培养，测序鉴定获得NOTCH1和eNOS基因敲除的细胞株。
进一步地，所述细胞株是NOTCH1单等位基因敲除和eNOS双等位基因敲除的细胞株。
本发明的有益效果为：
本发明用于NOTCH1和eNOS基因敲除的sgRNA及其应用，通过特定的sgRNA构建的CRISPR/Cas9***，对五指山猪耳成纤维细胞的NOTCH1和eNOS进行编辑，造成了片段缺失，导致移码敲除，获得了NOTCH1单等位基因编辑（即NOTCH1+/-）和eNOS双等位基因编辑（即eNOS-/-）的猪耳成纤维细胞，可用于制备“NOTCH1+/-和eNOS-/-”基因编辑猪，作为动脉粥样硬化动物模型用于医学和药物研发。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1 “NOTCH1+/-和eNOS-/-”猪胚胎图
具体实施方式
实施例1 NOTCH1、eNOS双基因编辑细胞系和应用研究
1.1设计构建靶向NOTCH1和eNOS基因的CRISPR/Cas9载体及验证其编辑效率
参照NCBI中猪NOTCH1（Gene ID: 110258061）和eNOS（Gene ID: 397557）基因序列信息，针对eNOS第三外显子和NOTCH1第三外显子分别设计多个靶向的sgRNA，合成sgRNA对应的两条（编码链和非编码链）短DNA序列并添加粘性末端，退火成双链(94 ℃, 10 min;37 ℃, 10 min，4℃，保存)。用BbsⅠ限制性内切酶对 PX458载体（带GFP荧光标记）做酶切回收，将酶切回收后的线性化载体与退火配对成双链DNA序列进行连接，进一步转化和涂板，经测序确认后，获得连接准确的CRISPR/Cas9载体（如下表1所示）。
利用0.1%的胰酶消化生长状态良好的五指山猪耳成纤维细胞（EF），1200rpm离心后，弃上清。用100μl电转液重悬细胞，每5×105个细胞混合单个CRISPR/Cas9 载体，混匀后转移至电转杯中，经Lonza核转染仪进行电转染，电击后的细胞接种至100mm 细胞培养皿中，用含有 20%FBS 的 DMEM 继续培养细胞48h。利用0.1%的胰酶消化将电转的细胞分别消化成单细胞悬液，经流式分选，分别收集带GFP绿色荧光的细胞。分别提取GFP阳性细胞的基因组DNA，进行PCR 扩增。分别在两个sgRNA作用位点两侧设计引物，如下：
SUS-eNOS-ID-F：5’- tcctgacttttgtgccacctgc-3’；
SUS-eNOS-ID-R：5’-caggaggaagtcatgggtgg-3’；产物大小为 410bp；
SUS- NOTCH1-ID-F:5’- gtgtttggcacaactgtgagg-3’；
SUS- NOTCH1-ID-R：5’- ccttaacggaccccaagcac-3’；产物大小为553bp；
CR 扩增体系如下：上、下游引物(10pmoL/μL)各 1μL；基因组 DNA 0.5μg，PremixLA Taq 10μL，灭菌蒸馏水补至 20μL。PCR 反应条件：95 ℃ 5 min；(95℃ 30 s, 53 ℃ 30s, 68℃ 20 s)×32cycles；68 ℃ 5 min；16 ℃保存。纯化回收 PCR 产物进行 TA 克隆，培养 12h 后，随机测序25个以上的单菌落，将测序结果与野生型基因序列进行比对，统计各个 sgRNA序列的编辑效率。结果如下表1所示。
表1“NOTCH1+/-和eNOS-/-”基因的sgRNA及其编辑效率
注：SEQ ID NO .1~7是sgRNA在载体上的核苷酸序列，SEQ ID NO .8 ~14是sgRNA在细胞内的核苷酸序列。
1.2 细胞转染筛选及单克隆鉴定
将有效编辑效率最高的eNOS-sgRNA6和NOTCH1-sgRNA3载体（带GFP荧光标记）共转染 EF，流式细胞仪富集带绿色荧光细胞，按100个细胞/每皿接种到100mm细胞培育皿中，培养 15d 左右，将单细胞克隆转入 48 孔细胞培养板中继续进行培养，待细胞增长到 80%~90%时，取部分细胞用于基因敲除鉴定。细胞裂解液法提取细胞基因组 DNA，PCR 测序分析基因敲除类型，将“NOTCH1+/-和eNOS-/-”单细胞克隆冻存，用于后续体细胞核移植实验。“NOTCH1+/-和eNOS-/-”单细胞克隆的基因型如表2所示。
表2 “NOTCH1+/-和eNOS-/-”单细胞克隆的基因型
由表2可见，被eNOS-sgRNA6和NOTCH1-sgRNA3载体转染的猪耳成纤维细胞获得了NOTCH1单等位基因（即NOTCH1+/-）和eNOS双等位基因敲除。
1.3 体外制备“NOTCH1+/-和eNOS-/-”猪胚胎
将“NOTCH1+/-和eNOS-/-”猪耳成纤维细胞，通过核移植的方法，注射到去核的体外成熟猪***内，经过融合、激活、体外培养。“NOTCH1+/-和eNOS-/-”重构胚胎制备如图1所示。
综上，本发明通过特定的sgRNA构建的CRISPR/Cas9***，对五指山猪耳成纤维细胞的NOTCH1和eNOS进行编辑，造成了片段缺失，导致移码敲除，获得了NOTCH1单等位基因编辑（即NOTCH1+/-）和eNOS双等位基因编辑（即eNOS-/-）的猪耳成纤维细胞，可用于制备“NOTCH1+/-和eNOS-/-”基因编辑猪，作为动脉粥样硬化动物模型用于医学和药物研发。
Claims (9)
1.一组sgRNA，其特征在于：它包括核苷酸序列如SEQ ID NO.10所示的eNOS-sgRNA6，和核苷酸序列如SEQ ID NO.13所示的NOTCH1-sgRNA3。
2.一组CRISPR/Cas9载体，其特征在于：它是连接有与eNOS-sgRNA6对应的双链DNA的载体，和连接有与NOTCH1-sgRNA3对应的双链DNA的载体；
所述eNOS-sgRNA6的核苷酸序列如SEQ ID NO.10所示；所述NOTCH1-sgRNA3的核苷酸序列如SEQ ID NO.13所示。
3.根据权利要求2所述的CRISPR/Cas9载体，其特征在于：所述载体为pX458质粒载体。
4.一种权利要求2或3所述CRISPR/Cas9载体的构建方法，其特征在于，它包括如下步骤：
取与eNOS-sgRNA6对应的双链DNA，***到pX458质粒载体中；取与NOTCH1-sgRNA3对应的双链DNA，***pX458质粒载体中，即得；
所述eNOS-sgRNA6的核苷酸序列如SEQ ID NO.10所示；所述NOTCH1-sgRNA3的核苷酸序列如SEQ ID NO.13所示。
5.权利要求2或3所述CRISPR/Cas9载体在制备NOTCH1和eNOS基因敲除的细胞株中的用途。
6.根据权利要求5所述的用途，其特征在于：所述细胞株是NOTCH1单等位基因敲除和eNOS双等位基因敲除的细胞株。
7.根据权利要求5所述的用途，其特征在于：所述细胞株包括五指山猪耳成纤维细胞。
8.一种NOTCH1和eNOS基因敲除的细胞株的构建方法，其特征在于：它包括如下步骤：
取权利要求2所述CRISPR/Cas9载体转染五指山猪耳成纤维细胞，富集带绿色荧光细胞培养，测序鉴定获得NOTCH1和eNOS基因敲除的细胞株。
9.根据权利要求8所述的构建方法，其特征在于：所述细胞株是NOTCH1单等位基因敲除和eNOS双等位基因敲除的细胞株。
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