CN114934073A - hoxa1a基因敲除斑马鱼突变体的构建方法和应用 - Google Patents
hoxa1a基因敲除斑马鱼突变体的构建方法和应用 Download PDFInfo
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Abstract
本发明公开了hoxa1a基因敲除斑马鱼突变体的构建方法和应用,通过CRISPR技术构建,依次包括设计gRNA位点—PCR扩增—gRNA的模板纯化—体外转录—gRNA纯化—显微注射—检测敲除效率—饲养至成鱼—与野生型交配—检测下一代胚胎是否携带突变位点—饲养成年后剪尾鉴定出杂合突变体—两杂合体交配得到纯合突变体的步骤。本发明首次利用CRISPR技术构建hoxa1a基因敲除斑马鱼突变体,其可作为动物模型用于研究hoxa1a基因在心脏发育过程中的作用以及为治疗Bosley‑Salih‑Alomainy综合征提供基础。
Description
技术领域
本发明属于分子生物学领域,具体涉及hoxa1a基因敲除斑马鱼突变体的构建方法和应用。
背景技术
CRISPR/Cas9***是规律成簇的间隔短回文重复(CRISPR)和CRISPR-associatedprotein9(Cas9)共同组成的***,是细菌用来防御噬菌体DNA注入和质粒转移的天然防御***。但现在被人类所重新利用,构建了很强的RNA引导的DNA靶向平台——主要用来基因组编辑、转录扰乱、表观遗传调控等。CRISPR/Cas9***是II型CRISPR***,它和其他***的区别在于,只是需要一个DNA内切酶Cas9来对与sgRNA20个互补碱基的带有PAM结构的DNA进行剪切,剪切后使DNA产生平末端的双链断裂(DSB),然后在进行非同源的末端连接过程中,容易随机***或者删除或者替换,或者进行高保真的同源定向修复DNA。CRISPR/Cas9***具有操作简单、效率高,成本低,毒性小等优势,受到了科研工作者的青睐,现在被广泛运用在不同物种中进行基因的定向改造。
hox基因是编码具有同源框的一大类转录因子家族,全名同源异型基因。hox基因无论片断大小,其序列中均含有一段由180-183个碱基组成的保守序列,该序列编码60-61个氨基酸所构成的多肽区域,称为同源结构域(hD)。转录因子hox基因的hD为DNA结合结构域,进而调控靶基因的表达。hox基因在早期胚胎发育身体形态构建过程中发挥着重要作用,其突变常常会导致身体相应部位产生缺陷,导致畸形。
先天性人类HOXA1综合征的Bosley-Salih-Alomainy综合征(BSAS)是由常染色体隐性截断HOXA1突变引起的。患者通常会出现以下症状:眼动、内耳空腔畸形、心脏畸形和发育迟缓,还有部分患者会出现从单侧颈内动脉发育不良到双侧发育不全的脑血管畸形。在小鼠中,Hoxa1作为一种同源域转录因子,对小鼠后脑菱形体的正常发育至关重要,而Hoxa1缺失的突变体会发生心血管畸形,Hoxa1缺失小鼠显示出诸如主动脉弓中断,锁骨下动脉异常和法洛四联症等缺陷,表明Hoxa1是心脏主动脉和流出道发育所必需的。目前,尚未发现利用CRISPR/Cas9技术构建hoxa1a基因敲除斑马鱼突变体鱼系相关报道。
发明内容
本发明的主要目的在于hoxa1a基因敲除斑马鱼突变体的构建方法,利用CRISPR技术设计一段特异性打靶hoxa1a的gRNA序列,通过显微注射的方式将斑马鱼中的hoxa1a基因敲除,获得可以稳定遗传的hoxa1a基因敲除斑马鱼突变体鱼系,
本发明的另一目的在于通过上述构建方法得到的hoxa1a基因敲除斑马鱼突变体的应用,hoxa1a突变体出现心脏发育缺陷等疾病相关表型,为研究hoxa1a及其同源基因在个体发育过程中的功能和致病机理提供重要的疾病模型。
本发明的目的是通过以下技术方案来实现的:
本发明提供hoxa1a基因敲除斑马鱼突变体的构建方法,通过CRISPR技术构建hoxa1a基因敲除斑马鱼突变体,依次包括设计gRNA位点—PCR扩增—gRNA的模板纯化—体外转录—gRNA纯化—显微注射—检测敲除效率—饲养至成鱼—与野生型交配—检测下一代胚胎是否携带突变位点—饲养成年后剪尾鉴定出杂合突变体—两杂合体交配得到纯合突变体的步骤,具体包括以下步骤:
(1)获取斑马鱼的hoxa1a基因序列;
(2)在斑马鱼hoxa1a基因的第一个外显子上设计如SEQ ID NO:1所示的靶点gRNA序列;
(3)设计并合成hoxa1a基因的gRNA引物F1和R1,序列分别如SEQ ID NO:2和SEQ IDNO:3所示;
(4)设计并合成hoxa1a基因的检测引物F2和R2,序列分别如SEQ ID NO:4和SEQ IDNO:5所示;
(5)使用步骤(3)的gRNA引物、gRNA骨架质粒为模板进行PCR扩增反应,电泳检测PCR产物后,纯化;
(6)上述PCR纯化产物在RNase-Free条件下进行体外转录得到gRNA,转录体系中加入T7聚合酶和NTP,37℃反应1.5h,纯化;
(7)将步骤(6)纯化后的gRNA和Cas9蛋白混合后显微注射到斑马鱼单细胞期的胚胎中,24h后取胚胎提取DNA,用上述F2/R2这对引物对敲除位点进行PCR扩增,用TAKARAT7E1酶进行酶切统计敲除效率,将敲除成功的小鱼饲养长大,作为F0;
(8)待F0斑马鱼性成熟后与野生型的斑马鱼杂交,得到杂合子,取胚胎提取DNA,并用F2/R2这对引物对敲除位点进行PCR扩增,用T7E1酶检测PCR产物突变效率并测序确认,将有突变的斑马鱼培养长大,作为F1;
(9)待F1斑马鱼性成熟后,将雌鱼和雄鱼的鱼尾切除进行尾鳍DNA提取,按照上述方法进行PCR扩增确认是否突变,测序确认后有突变的斑马鱼PCR产物和19T载体相连接,经过转化和蓝白斑筛选挑选白色菌落进行摇菌,通过菌液PCR鉴定并挑选阳性菌液,测序确认每条鱼的突变类型,具有相同突变类型的雌雄斑马鱼进行配对即得。
优选地,步骤(5)中,所述PCR反应条件为:预变性94℃3min,变性94℃30s,退火65℃30s,延伸72℃30s进行35个循环,再72℃10min,最后保温在12℃。
优选地,步骤(7)中,所述PCR反应条件为:预变性94℃3min,变性94℃30s,退火62℃30s,延伸72℃40s进行35个循环,再72℃10min,最后保温在12℃。
优选地,步骤(8)和(9)中,F0斑马鱼和F1斑马鱼性成熟的时间均为3-4个月。
优选地,步骤(7)中,所述T7E1酶切检测反应体系如下:PCR产物5μL、Buffer 2 1.1μL、ddh2O 4μL,反应条件为:95℃-30s,85℃-5min,4℃∞,反应后加0.25μL T7E1酶37℃水浴45min,电泳检测。
优选地,步骤(7)的显微注射中,Cas9蛋白的终浓度800ng/μL,gRNA的终浓度100ng/μL,注射量为1nL。
优选地,第3天时,hoxa1a-/-纯合突变体相较于野生型斑马鱼心室有变大现象并且心室心肌层变厚,一个月的幼鱼hoxa1a-/-纯合突变体相较于野生型出现心包腔肿大、流出道血管平滑肌增厚和心室心肌层增厚的异常表型。
本发明还提供通过上述hoxa1a基因敲除斑马鱼突变体的构建方法得到的hoxa1a基因敲除斑马鱼突变体在构建与hoxa1敲除综合征相关的动物模型和药物筛选中的应用。
与现有技术相比,本发明的有益效果在于:
一、本发明首次在斑马鱼中利用CRISPR/Cas9技术获得hoxa1a基因缺失突变体,hoxa1a可稳定遗传,方便对hoxa1a基因的功能进行深入研究。
二、本发明利用CRISPR/Cas9技术在hoxa1a设计一段独特的sgRNA序列使斑马鱼中hoxa1a基因被敲除,又不影响其他基因,形成hoxa1a基因特异敲除的斑马鱼。
三、本发明针对不同目的基因的靶点分别设计一对特异性引物,然后运用双外侧引物进行片段敲除检测和测序确认,成功构建hoxa1a基因缺失的斑马鱼突变体鱼系,可用于构建与hoxa1a基因相关疾病的动物模型,为后续研究hoxa1a基因在动物体生长发育的调节机制和治疗Bosley-Salih-Alomainy综合征等由hoxa1a引起的相关疾病起到重要作用。
四、本发明通过CRISPR/Cas9技术构建hoxa1a基因缺失的斑马鱼突变体,与传统的基因编辑技术相比具有毒性小、准确性高、效率高、成本低、易操作、成功周期短等特点,而相对于传统遗传学操作方法,CRISPR/Cas9***能对预编辑的目标序列进行剪切,具有很强的正选压力,不需要额外使用选择标记,避免了传统操作方法中常遇到的可用选择标计已有限、引入抗生素标记产生生物安全隐患等。
附图说明
图1是实施例中hoxa1a基因敲除的纯合斑马鱼突变体的筛选结果:(A)hoxa1a基因敲除的模式图;(B、C)F1代hoxa1a基因敲除的检测结果;(D)不同突变类型序列比对图;(E)杂合子和纯合子测序峰图。
图2是实施例中野生型和hoxa1a-/-发育3天的胚胎光镜图片和心脏石蜡切片:(A,B)心脏光镜图;(C,D)心脏石蜡切片图;相比于野生型,hoxa1a突变体心室变大,心室心肌层变厚;比例尺:10um。
图3是实施例中野生型和hoxa1a-/-一个月幼鱼心脏石蜡切片:(A,D)心室;(B,D)流出道;(C,F)心室心肌层;幼鱼心脏石蜡切片进一步证实hoxa1a基因敲除的突变体心室变大,流出道异常和心室心肌层变厚;比例尺:10um。
图4是实施例中野生型和hoxa1a-/-胚胎的心脏原位杂交:(A,B)cmlc2探针;(C,D)vmhc探针;(E,F)nppa探针;(G,H)nppb探针;使用心脏特异性标记基因cmlc2、vmhc、nppa、nppb探针进行原位杂交,发现斑马鱼hoxa1a-/-纯合突变体相较于野生型,突变体心室变大(图4A,B,C,D)、vmhc基因信号过量表达(图4C,D);原位杂交实验显示突变体的nppa、nppb探针在房室间隔指示处的表达异常,并且突变体出现心脏环化角度异常(图4E,F,G,H);比例尺:200um。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例的附图对本发明实施例的技术方案进行清楚、完整地描述。显然,所描述的实施例是本发明的一部分实施例,而不是全部的实施例。基于所描述的本发明的实施例,本领域普通技术人员在无需创造性劳动的前提下所获得的所有其它实施例,都属于本发明保护的范围。
以下实施例中所用的引物均由上海生工生物公司合成。gRNA骨架质粒的序列为TAATACGACTCACTATA+target sequence+GTTTTAGAGCTAGAAATAGC,来自于文献:Chang N,SunC,Gao L,Zhu D,Xu X,Zhu X,Xiong JW,Xi JJ.Genome editing with RNA-guided Cas9nuclease in zebrafish embryos,Cell Res,2013,23(4):465-472)。Cas9购自南京金斯瑞生物公司,PCR中反应的酶和19T载体购自北京全式金生物公司,酶切检测中使用的T7E1酶是由TARAKA生产的,其余无机(NaOh,Tris-hCl等)及有机试剂(乙醇等)购自国药集团化学试剂有限公司,本发明中使用的斑马鱼为野生型斑马鱼AB品系,购自中国科学研究院上海生命科学院生物化学与细胞生物学研究所。斑马鱼的hoxa1a基因序列在NCBI(https://www.ncbi.nlm.nih.gov/)网站上获取,使用CRISPick(https://portals.broadinstitute.org/gppx/crispick/public)在斑马鱼hoxa1a基因的第一个外显子上设计gRNA序列。
实施例1
本实施例通过CRISPR/Cas9技术得到hoxa1a基因敲除斑马鱼突变体,其技术路线为:设计gRNA位点—PCR扩增—gRNA的模板纯化—体外转录—gRNA纯化—显微注射—检测敲除效率—饲养至成鱼—与野生型交配—检测下一代胚胎是否携带突变位点—饲养成年后剪尾鉴定出杂合突变体—两杂合体交配得到纯合突变体,主要包括如下步骤:
(1)在hoxa1a基因的第一个外显子上设计gRNA序列:
5’-GGTGGAGATGGAGGTAGCGG-3’(SEQ ID NO:1)。
(2)设计并合成gRNA引物,引物序列为:F1:5’-TAATACGACTCACTATAGGTGGAGATGGAGGTAGCGGGTTTTAGAGCTAGAAATAGC-3’(SEQ ID NO:2);R1:5’-AAAAAAAGCACCGACTCGGTGCCAC-3’(SEQ ID NO:3)。
(3)以上述的gRNA引物、gRNA骨架质粒为模板进行PCR反应,反应体系为:
PCR反应条件为:预变性94℃3min;变性94℃30s,退火65℃30s,延伸72℃30s进行35个循环,再72℃10min,最后保温在12℃;电泳检测PCR产物后,利用DNA纯化试剂盒进行纯化,用RNase-Free的水进行溶解洗脱。
(4)RNase-Free条件下,将上述的PCR纯化产物进行体外转录得到gRNA,转录体系为:
反应条件为:37℃-1.5h,后加入DNase 1μL,37℃-15min。体外转录后用LiCl沉淀法对gRNA进行纯化。具体方法:向上述反应液中加入2.5μL的4M LiCl2,再加入100μL的100%乙醇;放置-80℃冰箱孵育至少2小时(也可以过夜处理);4℃、12000rpm、15min,弃上清;用预冷的70%乙醇洗两次,4℃、8000rpm、10min,弃上清,目的是为了去除杂质;超净台中室温通风晾干5min;最后加入15μL RNase-free水溶解,Nanodrop检测浓度和电泳检测。
(5)将前述纯化后的gRNA和Cas9蛋白混合后显微注射到斑马鱼单细胞期的胚胎中,注射时Cas9蛋白终浓度800ng/μL,gRNA终浓度100ng/μL,注射1nL。24h后取3组,5枚胚胎一组放入PCR小管中,利用碱裂法提取DNA:向每管中加入30μL 50mM NaOh溶液,95℃-10min后取出,在震荡仪上充分震碎组织后;继续95℃-10min;加入3μL 1M Tris-hCl(pH=8)后震荡混匀离心10000rpm-5min。
设计检测引物:
F2:5’-TATCACTAGCGCCCGAACAC-3’(SEQ ID NO:4),
R2:5’-TCACAGACGATTCCACGTCC-3(SEQ ID NO:5)。
用上述引物对敲除位点进行PCR扩增,反应体系为:
PCR反应条件为:预变性94℃3min;变性94℃30s,退火62℃30s,延伸72℃40s进行35个循环,再72℃10min,最后保温在12℃;后用TAKARA T7E1酶进行敲除效率检测(图1),将敲除成功的那批注射组小鱼饲养长大,作为F0;
其中,T7E1酶切检测反应体系如下:
反应条件为:95℃-30s,85℃-5min,4℃∞,反应后加0.25μL T7E1酶37℃水浴45min,电泳检测。
(6)3-4个月后F0斑马鱼性成熟,将突变的斑马鱼与野生型的斑马鱼杂交,得到一定概率的杂合子,取胚胎按上述碱裂法进行DNA提取,并用上述引物对敲除位点进行PCR扩增后,用T7E1酶检测突变效率(图1,B,C),并送测序确认,将有突变的斑马鱼培养长大;作为F1。
(7)3-4个月后F1斑马鱼性成熟,将雌鱼和雄鱼的鱼尾切除进行尾鳍DNA提取,PCR和T7E1酶切确认是否突变,并通过测序确认(图1,E)。将测序后发现突变的斑马鱼的PCR产物和19T载体相连接后,经过转化和蓝白斑筛选后,挑选白色菌落进行摇菌,通过菌液PCR鉴定后,挑选阳性菌液送测序,确定每条鱼的突变类型;将相同突变类型的雌雄斑马鱼进行配对,从而得到敲除hoxa1a的纯合突变体。
将实施例1制备的hoxa1a的纯合突变体自交得到纯合后代,通过对hoxa1a基因敲除纯合突变体进行观察发现,第3天时,斑马鱼hoxa1a-/-纯合突变体相较于野生型斑马鱼,其心室有初步变大现象并且心室心肌层变厚(图2);通过对一个月的幼鱼进行心脏石蜡切片及HE染色实验,发现斑马鱼hoxa1a-/-纯合突变体相较于野生型,出现心包腔肿大、流出道血管平滑肌增厚和心室心肌层增厚的异常表型。(图3)。
将实施例1制备的hoxa1a的纯合突变体自交得到纯合后代,使用心脏特异性标记基因cmlc2、vmhc、nppa、nppb探针进行原位杂交,发现斑马鱼hoxa1a-/-纯合突变体相较于野生型,突变体心室变大(图4A,B,C,D)、vmhc基因信号过量表达(图4C,D);原位杂交实验显示突变体的nppa、nppb探针在房室间隔指示处的表达异常,并且突变体出现心脏环化角度异常(图4E,F,G,H)。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 上海海洋大学
<120> hoxa1a基因敲除斑马鱼突变体的构建方法和应用
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Claims (8)
1.hoxa1a基因敲除斑马鱼突变体的构建方法,其特征在于,通过CRISPR技术构建,依次包括设计gRNA位点—PCR扩增—gRNA的模板纯化—体外转录—gRNA纯化—显微注射—检测敲除效率—饲养至成鱼—与野生型交配—检测下一代胚胎是否携带突变位点—饲养成年后剪尾鉴定出杂合突变体—两杂合体交配得到纯合突变体的步骤,具体包括以下步骤:
(1)获取斑马鱼的hoxa1a基因序列;
(2)在斑马鱼hoxa1a基因的第一个外显子上设计如SEQ ID NO:1所示的靶点gRNA序列;
(3)设计并合成hoxa1a基因的gRNA引物F1和R1,序列分别如SEQ ID NO:2和SEQ ID NO:3所示;
(4)设计并合成hoxa1a基因的检测引物F2和R2,序列分别如SEQ ID NO:4和SEQ ID NO:5所示;
(5)使用步骤(3)的gRNA引物、gRNA骨架质粒为模板进行PCR扩增反应,电泳检测PCR产物后,纯化;
(6)上述PCR纯化产物在RNase-Free条件下进行体外转录得到gRNA,转录体系中加入T7聚合酶和NTP于37℃反应1.5h,纯化;
(7)将步骤(6)纯化后的gRNA和Cas9蛋白混合后显微注射到斑马鱼单细胞期的胚胎中,24h后取胚胎提取DNA,用上述F2/R2这对引物对敲除位点进行PCR扩增,用TAKARA T7E1酶进行酶切统计敲除效率,将敲除成功的小鱼饲养长大,作为F0;
(8)待F0斑马鱼性成熟后与野生型的斑马鱼杂交,得到杂合子,取胚胎提取DNA,并用F2/R2这对引物对敲除位点进行PCR扩增,用T7E1酶检测PCR产物突变效率并测序确认,将有突变的斑马鱼培养长大,作为F1;
(9)待F1斑马鱼性成熟后,将雌鱼和雄鱼的鱼尾切除进行尾鳍DNA提取,按照上述方法进行PCR扩增确认是否突变,测序确认后有突变的斑马鱼PCR产物和19T载体相连接,经过转化和蓝白斑筛选挑选白色菌落进行摇菌,通过菌液PCR鉴定并挑选阳性菌液,测序确认每条鱼的突变类型,具有相同突变类型的雌雄斑马鱼进行配对即得。
2.根据权利要求1所述hoxa1a基因敲除斑马鱼突变体的构建方法,其特征在于,步骤(5)中,所述PCR反应条件为:预变性94℃3min,变性94℃30s,退火65℃30s,延伸72℃30s进行35个循环,再72℃10min,最后保温在12℃。
3.根据权利要求1所述hoxa1a基因敲除斑马鱼突变体的构建方法,其特征在于,步骤(7)中,所述PCR反应条件为:预变性94℃3min,变性94℃30s,退火62℃30s,延伸72℃40s进行35个循环,再72℃10min,最后保温在12℃。
4.根据权利要求1所述hoxa1a基因敲除斑马鱼突变体的构建方法,其特征在于,步骤(8)和(9)中,F0斑马鱼和F1斑马鱼性成熟的时间均为3-4个月。
5.根据权利要求1所述hoxa1a基因敲除斑马鱼突变体的构建方法,其特征在于,步骤(7)中,所述T7E1酶切检测反应体系如下:PCR产物5μL、Buffer 2 1.1μL、ddh2O 4μL,反应条件为:95℃-30s,85℃-5min,4℃∞,反应后加0.25μL T7E1酶37℃水浴45min,电泳检测。
6.根据权利要求1所述hoxa1a基因敲除斑马鱼突变体的构建方法,其特征在于,步骤(7)的显微注射中,Cas9蛋白的终浓度800ng/μL,gRNA的终浓度100ng/μL,注射量为1nL。
7.根据权利要求1所述hoxa1a基因敲除斑马鱼突变体的构建方法,其特征在于,hoxa1a-/-纯合突变体第3天时心室有变大现象并且心室心肌层变厚,一个月的hoxa1a-/-纯合突变体出现心包腔肿大、流出道血管平滑肌增厚和心室心肌层增厚的异常表型。
8.hoxa1a基因敲除斑马鱼突变体在构建与hoxa1基因敲除综合征相关的动物模型和药物筛选中的应用,所述hoxa1a基因敲除斑马鱼突变体通过权利要求1至7任一项所述hoxa1a基因敲除斑马鱼突变体的构建方法得到。
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