CN116064559A - nomo基因缺失型斑马鱼的构建方法 - Google Patents
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Abstract
本发明涉及基因工程技术领域,尤其涉及nomo基因缺失型斑马鱼的构建方法。通过CRISPR/Cas9基因编辑技术,在斑马鱼的nomo基因上设计合适的打靶位点,构建了nomo基因缺失型斑马鱼模型。构建获得的斑马鱼模型中nomo基因表达发生改变,有助于进一步揭示原发性纤毛运动障碍(Primary ciliary dyskinesia,PCD)特异表型——心脏左右非对称发育的整个过程以及调控这些过程的分子机制,在医学上对PCD及左右非对称发育病理生理的理解和新的治疗方案的研发中具有十分重要的意义。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及nomo基因突变型斑马鱼的构建方法。
背景技术
NOMO1(OMIM:617824),又名NODAL Modulator 1,基因定位于人染色体16p13.11区带,编码1222个氨基酸,分子量约130KD,该基因在人类胚胎早期的多个组织中都有表达,特别的是,该基因与左右不对称发育密切相关。有研究表明NOMO1编码的蛋白与Nicalin结合形成的复合蛋白,为内质网驻留的转膜蛋白,该复合物在一定程度上抑制Nodal信号通路,而Nodal信号通路在心脏非对称发育及其他器官形成中至关重要。
斑马鱼与人类骨骼发育过程中的基因、信号通路有高度同源性,且nomo(人类NOMO1基因在斑马鱼中对应的同源基因为nomo)基因进化上较为保守,研究发现nomo在斑马鱼胚胎中早期表达量特别高。而且,与其他动物模型相比,斑马鱼具有个体小、易于饲养、发育快、繁殖能力强、体外受精、胚胎体外发育且透明等优点。并且斑马鱼早期发育阶段可不依赖有缺陷的心血管***,因此能活体观察心血管***缺陷的突变体,斑马鱼心脏与人类心脏发育相似的基因调控图解及心电生理学。通过CRISPR/Cas9基因编辑技术,干扰掉斑马鱼体内的nomo基因,并且通过遗传学手段研究其功能,有助于进一步揭示原发性纤毛运动障碍(Primary ciliary dyskinesia,PCD)特异表型——心脏左右不对称发育的整个过程以及调控这些过程的分子机制,在医学上对PCD病理的理解和新的治疗方案的研发中具有十分重要的意义。
基因打靶技术起源于20世纪80年代末,是一种通过对基因组进行定点修饰来研究基因功能的重要方法手段,也可用于治疗人类的各种遗传性疾病。该技术主要是利用缺失突变、基因灭活、染色体大片段删除以及外源基因导入等方式来改变生物的遗传信息,并且在生殖系中稳定遗传后表达突变性状,从而研究生物体内特定基因在生长发育过程中的作用,所以这类技术手段已成为现代分子生物学研究热点。传统的基因打靶技术是建立在胚胎干细胞(ESC)和同源重组技术的基础之上,故打靶技术效率极低。2013年初,一种全新的人工核酸内切酶clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)9,能更高效且更精确地在生物体基因组中突变特定基因,且制作简单、成本低,且可同时对靶基因上多个位点进行剪切,突变任意数目的单个基因,但同时该技术存在一定的缺陷,其脱靶率相对较高。
发明内容
有鉴于此,本发明要解决的技术问题在于提供nomo基因突变型斑马鱼的构建方法。本发明找到了合适的打靶位点,通过CRISPR/Cas9基因编辑技术,选育出nomo基因突变型斑马鱼。
参见图1,图1为本发明提供的CRISPR/Cas9打靶***原理图,其通过cas9和gRNA对基因靶位点进行敲除,靶位点的选择会影响基因敲除效率。
本发明提供了斑马鱼nomo基因的靶位点,其具有如SEQ ID NO:1所示的核酸序列,具体为:GGGACCTCTACGCTCAAATGAGG。参见图2,图2是nomo基因上靶位点的结构图,该靶位点位于19号外显子结构域,更有利于基因的敲除。
本发明在前期进行了大量实验,结果表明将靶位点设置在SEQ ID NO:1所示的核酸序列结构域,更有利于基因的敲除。并且,本发明中具体靶点片段的选择更合理,从而进一步降低脱靶效应。
本发明还提供了扩增靶位点的引物对,其包括如SEQ ID NO:2、SEQ ID NO:3所示的核酸序列的两条引物,具体为:
F(5’-TCTCACCATTGTGTGTTCAT-3’)
R(5’-TCTTTGGATGAAGGTGTGAC-3’)。
本发明还提供了靶向所述靶位点的gRNA,其具有如SEQ ID NO:4所示的核酸序列。一些实施例中,所述靶向靶位点的gRNA的核酸序列具体为:GGGACCUCUACGCUCAAAUG。
本发明还提供了重组载体,其包括骨架载体和所述的gRNA片段。
本发明还提供了敲除斑马鱼nomo基因的试剂,其包括:Cas9蛋白和本发明所述的gRNA。
本发明所述的试剂中,其包括水和5ng/μL Cas9蛋白、30~40ng/μLgRNA。
本发明还提供了nomo基因缺失型斑马鱼的构建方法,其以本发明所述的试剂,敲除斑马鱼受精卵中的nomo基因。
本发明实施例中,所述斑马鱼受精卵为受精0.5h内的斑马鱼受精卵。
本发明实施例中,所述试剂的用量为每个斑马鱼受精卵1.8nL试剂。
本发明所述的方法中,还包括筛选的步骤,所述筛选采用PCR和测序。
通过CRISPR/Cas9基因编辑技术,在斑马鱼的nomo基因上设计合适的打靶位点,在体外合成特异性sgRNA和Cas9-mRNA,混合后显微共注射至斑马鱼一细胞内,胚胎培养48h后,通过选取胚胎进行基因型分析,从而证实了所选位点的有效性。本发明提供选定的靶位点,构建了nomo基因缺失型斑马鱼模型,构建获得的斑马鱼模型中nomo基因表达发生改变,有助于进一步揭示原发性纤毛运动障碍(Primary ciliary dyskinesia,PCD)特异表型—心脏左右非对称发育的整个过程以及调控这些过程的分子机制,在医学上对PCD及左右非对称发育病理生理的理解和新的治疗方案的研发中具有十分重要的意义。
附图说明
图1是CRISPR/Cas9打靶***原理图;
图2是nomo基因上靶位点的结构图;
图3是斑马鱼F1代杂合突变体测序图;
图4是F2代缺失型与WT基因型测序比对图。
具体实施方式
本发明提供了nomo基因突变型斑马鱼的构建方法,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
通过CRISPR/Cas9基因编辑技术,在斑马鱼的nomo基因上设计合适的打靶位点,在体外合成的特异性sgRNA和Cas9蛋白,显微共注射进入斑马鱼一细胞内,胚胎培养50h后,通过选取胚胎进行基因型分析,鉴定所设打靶位点的有效性。本发明能更高效且更精确地在生物体基因组中突变特定基因,且制作简单、成本低,且可同时对靶基因上多个位点进行剪切,突变任意数目的单个基因。且干扰掉nomo基因,并且通过遗传学手段研究其功能,有助于进一步揭示心脏发育的整个过程以及调控这些过程的分子机制,在医学上心脏疾病病理的理解和新的治疗方案的研发中具有十分重要的意义。本发明采用的试材皆为普通市售品,皆可于市场购得。
下面结合实施例,进一步阐述本发明:
实施例1
1)分别设计CRISPR/Cas9基因敲除靶位点和检测引物
在National Center for Biotechnology Information(NCBI)上查询斑马鱼nomo基因的基因组DNA序列,在网站SMART上分析其功能结构域,根据CRISPR/Cas敲除原理,设计nomo基因的靶位点;靶位点的选择遵循以下标准:5’-GG-(N)18-NGG-3’;其中5’端的GG二核苷酸是T7启动子的一部分,保证靶位点的3’端是NGG;靶点的选择位置在基因的结构域内。
靶位点序列为:GGGACCTCTACGCTCAAATGAGG。
上述特异性靶位点PCR引物如下:
PCR检测引物(PCR检测引物上下游引物分别位于18号内含子以及19号内含子上)
F(5’-TCTCACCATTGTGTGTTCAT-3’)
R(5’-TCTTTGGATGAAGGTGTGAC-3’)
2)构建gRNA表达载体以及特异性gRNA体外合成
以下转录实验中所用Tip头、EP管均为DEPC处理过的RNase-Free。
a.首先将gRNA骨架克隆到P42250载体上;
b.特异性gRNA体外合成BsaI限制性内切酶线性化此质粒,酶切反应总体积为20ul,体系反应如下:
混匀后于37℃水浴,酶切4h以上;
c.以线性化的p42250载体为模板,通过下述特异性引物进行PCR,扩增出用于特异性gRNA合成的双链DNA;
正向特异性靶位点引物F:T7启动子+20pb靶序列+20bp sgRNA上游骨架;
反向引物R:20bp sgRNA下游骨架;
PCR反应体系(50μL)如下:
震荡混匀之后,4℃离心,于PCR仪上进行扩增反应,反应条件为:预变性95℃5min,再重复33次循环以下步骤:变性95℃30s—退火;55℃30s—延伸72℃30s,再72℃8min;反应结束后,离心PCR产物,取1μL样品点样于1.8%琼脂糖凝胶上进行电泳,凝胶成像***拍摄结果;
d.检测确定条带正确之后,进行琼脂糖凝胶DNA回收,纯化回收PCR产物;
e.测定纯化的DNA浓度,再以此DNA为模板,用20μL体系进行体外转录,合成特异性gRNA;具体步骤如下:
体外转录反应体系(20μL):
将反应物都加入1.5mL RNase-Free的EP管中,混匀之后,放置于37℃水浴锅中反应2.5h,向转录体系中加入1μL DNA酶,放置于37℃水浴锅中反应30min,用以消化DNA模板,然后取1μL的gRNA样品,进行琼脂糖凝胶电泳,以检测转录结果,若转录产物大小与预期的相符,则说明转录成功;
f.特异性gRNA的纯化
用RNeasy Mini kit试剂盒纯化转录成功的gRNA,保存于-20℃;取纯化之后的gRNA溶液1μL测定浓度;
gRNA的具体序列为:GGGACCUCUACGCUCAAAUG。
3)斑马鱼胚胎的显微注射
按常规方法收取斑马鱼受精卵,在受精后30min之内,用吸管吸取胚胎转移至用琼脂糖制作的显微注射专用培养皿中。在进行显微注射之前,将Cas9 mRNA和上述gRNA配成混合液,充分混匀,使Cas9蛋白的终浓度为5ng/μL,gRNA的终浓度为30~40ng/μL,注射约1.8nL Cas9蛋白和gRNA混合液于一细胞期的受精卵内;注射过的受精卵放置于E3水中,28℃孵化;在体式显微镜下观察胚胎表型,筛选正常发育的胚胎用于靶位点突变分析;
显微注射体系如下:
其中,E3水为5mmol/L NaCl,0.33mmol/L CaCl2,0.33mmol/L MgSO4,0.17mmol/LKCl的混合物。
4)Sanger测序检测靶位点的有效性
对斑马鱼胚胎进行显微注射之后,挑选部分发育正常的5~10个早期胚胎,检测其nomo基因是否存在突变,有助于验证靶位点的选择是否有效果,显微注射操作是否规范;
a.提取斑马鱼基因组
斑马鱼胚胎受精48小时后(48hpf),分别收集野生型和注射后胚胎于1.5mL Ep管中,每管5~10颗胚胎,按照下述方法提取基因组DNA,具体步骤如下:
向装有胚胎的Ep管中加入200μL细胞裂解液,2μL蛋白酶K,放置于55℃水浴锅中裂解过夜;
裂解完成后,放在振荡器上充分震荡,加入等体积预先冷却的异丙醇于Ep管中,充分颠倒混匀,于4℃条件下,12000rpm离心10min,倒掉上清液;
加入10μL去离子水,充分吹打混匀,琼脂糖凝胶电泳检测提取效率;
b.PCR扩增目的序列
提取基因组DNA后,根据CRISPR靶位点上下游约100~250bp的基因组区域,利用Primer Premier 5.0软件设计引物序列以扩增出目的DNA片段;
PCR反应体系(20μL)如下:
震荡混匀之后,4℃离心,于PCR仪上进行扩增反应;反应条件如下:反应条件为:预变性95℃5min,再重复33次循环以下步骤:变性95℃30s—退火55℃30s—延伸72℃30s,再72℃8min;
c.用1.8%琼脂糖凝胶电泳检测PCR产物;
d.送部分纯化之后的目的DNA片段进行Sanger测序,由测序的峰图来初步获得***或缺失的信息;
e.注射两个月之后,进行剪尾鉴定,同上鉴定步骤;
5)目的序列的TA克隆
对PCR初步鉴定有突变可能的目的序列再进行Sanger测序,若测序峰图有双峰,并且测序结果显示有***或缺失现象的目的序列,接下来进行TA克隆之后挑取单克隆作进一步检测;
6)菌液的Sanger测序
将菌液PCR结果显示条带大小符合预期结果的菌液送往测序,根据测序之后给出的峰图和序列,在NCBI上与标准目的序列进行对比,根据比对结果,分析出每个单克隆的突变类型;
7)获得可遗传的斑马鱼突变体的F1代
通过前面一系列筛选确定了斑马鱼突变体F0代,紧接着将F0代突变体分别与野生型斑马鱼杂交得到F1代胚胎,置于28℃培养,初期观察F1代存活率发育情况。受精两天后,每个突变体F1代分别取5~10个胚胎进行突变遗传性鉴定。将每个胚胎单独提取基因组,进行Sanger测序,如果此突变可以遗传到F1代,测序峰图有双峰;将斑马鱼突变体的F1代养大至2~3个月,分别对每条F1代斑马鱼成鱼进行剪尾,PCR、TA克隆等步骤,筛选F1代突变体(具体方法如前面所述);
参见图3,图3是斑马鱼F1代杂合突变体测序图,由图3可知,斑马鱼F1代杂合突变体出现了8bp的杂合缺失,箭头指示自175bp处出现-8bp杂合缺失。
8)获得斑马鱼突变体的F2代纯合子
从F1代突变体筛选相同突变的雌鱼和雄鱼,杂交得到F2代,置于28℃,受精四天后取部分胚胎进行鉴定。将每个胚胎单独提取基因组,通过PCR扩增分析并测序,初步检验是否可以得到nomo突变体纯合子。如检验结果证明存在纯合子,则养大后再单条剪尾鉴定。
参见图4,图4是F2代缺失型与WT基因型测序比对图,由图4可知,nomo基因一共有8个碱基的纯合缺失(前面7个碱基,后面1个碱基,共8个碱基造成移码)。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.斑马鱼nomo基因的靶位点,其具有如SEQ ID NO:1所示的核酸序列。
2.权利要求1所述靶位点的扩增引物,其包括如SEQ ID NO:2、SEQ ID NO:3所示的核酸序列的两条引物。
3.靶向权利要求1所述靶位点的gRNA,其具有如SEQ ID NO:4所示的核酸序列。
4.重组载体,其包括骨架载体和权利要求3所述的gRNA片段。
5.敲除斑马鱼nomo基因的试剂,其包括:Cas9蛋白和权利要求3所述的gRNA。
6.根据权利要求5所述的试剂,其特征在于,其包括水和5ng/μL Cas9蛋白、30~40ng/μL gRNA。
7.nomo基因缺失型斑马鱼的构建方法,其特征在于,以权利要求5或6所述的试剂,敲除斑马鱼受精卵中的nomo基因。
8.根据权利要求7所述的构建方法,其特征在于,所述斑马鱼受精卵为受精0.5h内的斑马鱼受精卵。
9.根据权利要求7或8所述的构建方法,其特征在于,所述试剂的用量为每个斑马鱼受精卵1.8nL试剂。
10.根据权利要求7或8所述的构建方法,其特征在于,还包括筛选的步骤,所述筛选采用PCR和测序。
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