CN107974466B - 一种鲟鱼CRISPR/Cas9基因编辑方法 - Google Patents
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Abstract
本发明提供了一种鲟鱼CRISPR/Cas9基因编辑方法，是将Cas9蛋白与靶基因gRNA混合后，利用显微注射的方式，注射到受精时间不超过20分钟的鲟鱼1细胞期受精卵的动物极的受精孔中。将100 ng/µL Cas9 nuclease蛋白与30 ng/µL gRNA混合，采用显微注射方法，将混合物导入小体鲟1细胞期受精胚的动物极，孵化后可获得靶基因突变率高达83.1%的小体鲟胚胎，从而建立小体鲟靶基因精准编辑技术。靶基因发生突变的小体鲟胚胎可通过PAGE方法筛选获得。该技术既可以用于研究鲟鱼基因的功能，又可以用于精准修饰鲟鱼内源靶基因，培育遗传改良的鲟鱼养殖新品系。
Description
技术领域
本发明涉及水产动物遗传育种和功能基因验证研究的技术领域，更具体涉及一种鲟鱼CRISPR/Cas9基因编辑方法。
背景技术
鲟鱼在生物系统演化方面处于软骨鱼和硬骨鱼之间的过渡时期，具有多倍化的基因组，在研究鱼类基因功能及演化方面具有重要的研究价值。但是，在鲟鱼中开展基因功能研究，现有的报道仅局限于基因克隆和表达模式分析(Abdolahnejad等，2015.Dong等，2015.Fajkowska等，2016.Song等，2016.Yarmohammadi等，2017)。鲟形目鱼类属大型经济鱼类。鲟鱼不仅肉质鲜美，营养全面，富含多种人体必需氨基酸和不饱和脂肪酸，而且是重要的鱼子酱生产材料(Simeanu等，2012)。但是，由于人类过度捕捞及水生态环境的破坏，野生鲟鱼资源急剧下降，很多鲟鱼甚至处于濒危状态(Billard等，2000)。
开展鲟鱼的遗传育种，培育鲟鱼养殖品种，是满足人类对鲟鱼产品日益增长的需求，保护野生鲟鱼资源的关键。但是，鲟鱼的基因功能研究和遗传改良育种一直受制于缺乏有效的技术手段。
基因编辑技术是利用靶向性的序列特异核酸酶对基因组进行定点突变或精准修饰的技术，包括锌指核酸酶ZFN、TALE核酸酶和CRISPR/Cas9系统。基因编辑技术在基因功能研究与经济物种的遗传改良中得到了广泛应用。迄今已在斑马鱼、青鱂等模式鱼类和罗非鱼、虹鳟、鲤鱼、沟鲶、野鲮、半滑舌鳎和黄鳝等经济鱼类中建立了高效的基因编辑技术(Doyon et al,2008；Ansai et al,2013；Qiu et al,2014；Li et al,2014；Yano et al,2014；Zhong et al,2016；Qin et al,2016；Chakrapani et al,2016；Cui et al,2017；Feng et al,2017)。但是，迄今没有在鲟鱼中建立基因编辑技术。
因此，建立鲟鱼基因编辑技术，一方面可以用于鲟鱼功能基因研究；另一方面可以用于探索研制鲟鱼养殖新品系，为养殖鲟鱼提供遗传改良的优良品种，保护鲟鱼野生资源。
发明内容
本发明提供了一种鲟鱼CRISPR/Cas9基因编辑的方法，通过构建靶基因的规律成簇间隔短回文重复(Clustered regularly interspaced short palindromic repeats/associated nuclease9,CRISPRs/Cas9)基因编辑载体，体外转录合成靶基因的gRNA，将Cas9nuclease蛋白与靶基因gRNA混合，采用显微注射方法，将混合物导入受精时间不超过20分钟的鲟鱼1细胞期受精卵的动物极受精孔中，孵化后可获得靶基因突变的鲟鱼。
为了达到上述的目的，本发明采用以下技术措施：
一种鲟鱼CRISPR/Cas9基因编辑的方法，包括：将Cas9蛋白与靶基因gRNA混合后，利用显微注射的方式，注射到受精时间不超过20分钟的鲟鱼1细胞期受精卵的动物极的受精孔中；
以上所述的方法中，优选的，所述的鲟鱼包括但不限于小体鲟，西伯利亚鲟(A.baerii)、施氏鲟(A.schrenckii)、俄罗斯鲟(A.gueldenstaedtii)；
以上所述的方法中，优选的，Cas9蛋白与靶基因gRNA的终浓度分别为100ng/μL和30ng/μL；
以上所述的方法中，优选的，每个受精卵注射体积为2nL。
与现有技术相比，本发明具有以下优点：
目前没有任何进行鲟鱼胚胎的显微操作技术，更没有进行鲟鱼内源基因精准编辑的技术。本发明第一次建立鲟鱼内源基因精准编辑技术，为开展鲟鱼基因的功能研究和鲟鱼养殖品种的遗传改良提供了强有力的技术手段。
现有的用于基因编辑的规律成簇间隔短回文重复(Clustered regularlyinterspaced short palindromic repeats/associated nuclease 9,CRISPRs/Cas9)系统，通常采用共注射Cas9mRNA和guide RNA(gRNA)的方式。由于Cas9mRNA进入细胞后需被翻译成蛋白继而发挥功能，因此Cas9mRNA在细胞内的翻译效率会影响核酸内切酶的突变效率。本发明采用直接注射Cas9蛋白的方式，省却了mRNA翻译的过程，提高了Cas9核酸内切酶对靶位点的切割频率。只需构建靶基因如ntl的gRNA载体，并体外转录合成gRNA。将Cas9蛋白和针对基因靶位点的gRNA按100ng/μL和30ng/μL的浓度(终浓度)混合后，采用显微注射方法导入小体鲟1细胞期受精卵中，存活率为93.9％，可以获得靶基因突变率高达83.1％的小体鲟胚胎，从而建立小体鲟靶基因精准编辑技术。靶基因发生突变的小体鲟胚胎可通过PAGE方法筛选获得。该技术既可以用于研究鲟鱼基因的功能，又可以用于精准修饰鲟鱼内源靶基因，培育遗传改良的鲟鱼养殖新品系。
附图说明
图1为小体鲟ntl基因第一外显子部分序列及gRNA靶位点；
下划线序列为靶点，黑色三角指示Cas9酶切开DNA双链的位置。
图2为小体鲟1细胞期受精卵的动物极受精孔；
箭头指示为动物极受精孔。
图3为小体鲟ntl基因突变的检测示意图；
图3中a：7个注射胚胎的靶位点突变率检测；Hm对应条带代表未发生突变的靶位点DNA片段，Ht对应条带代表碱基发生减少、增加或改变的靶位点DNA片段；
图3中b：根据Ht条带和Hm条带亮度计算得出的靶位点突变频率；
图3中c：第3和第4枚检测胚胎的外形，可见脊柱弯曲、尾部变短。
具体实施方式：
本发明所述技术方案，如未特别说明，均为本领域的常规方案，所述试剂或材料，如未特别说明，均来源于商业渠道。本发明以编辑小体鲟ntl基因为例，建立了鲟鱼内源基因精准的基因编辑技术，该技术可以用于研究鲟鱼基因的功能和鲟鱼品种的遗传改良。
实施例1：
一种鲟鱼CRISPR/Cas9基因编辑的方法，包括下述步骤：
1)小体鲟1细胞期受精卵的获得：
挑选性成熟的小体鲟亲鱼，在室内养殖系统中暂养。距人工繁殖36小时前，注射LHR Ha和DOM混合物进行人工催产，注射剂量为10μg LHRHa+1mg DOM/kg体重，每隔12个小时注射1次，雌性鲟鱼连续注射2次，雄性鲟鱼注射1次。待亲鱼有排精/排卵迹象，挤出精液到干燥的烧杯，挤出卵到玻璃平皿。用0.3×Danieau buffer[17mM NaCl，2m M KCl，0.12mMMgSO4，1.8mM Ca(NO3)2，1.5mM HEPES，pH 7.6]将精液稀释100倍，进行人工授精。受精时间1分钟，随后洗去多余的精液，即获得可用于显微注射的小体鲟受精卵。
2)构建小体鲟靶基因(ntl)的guide RNA：
针对小体鲟的靶基因ntl(Genebank MG520324)，利用设计网站(http://zifit.partners.org/ZiFiT/)设计gRNA靶点，为GGCTTGAAGACGTGGATCTT(图1)。按照文献[Hwang WY,Fu Y,Reyon D,Maeder ML,Tsai SQ,Sander JD,Peterson RT,Yeh JR,JoungJK(2013)Efficient genome editing in zebrafish using a CRISPR-Cas system.NatBiotechnol 31:227-229]所述方法合成guide RNA(gRNA)。具体步骤如下：合成两条Oligo序列，F1-TAG GCTTGAAGACGTGGATCTT，R1-AAACAAGATCCACGTCTTCAAG。F1和R1引物各以10mM混合，先置于95℃5分钟，然后缓慢降温至37℃。DR274质粒(Addgene plasmid 42250)用BsaI(NEB,R0535)酶切后，与退火产物连接。将连接产物转化入感受态大肠杆菌。用引物M13(-47)和引物F1做PCR以挑出阳性克隆。PCR反应条件为：95℃2分钟，95℃15秒，56℃15秒，72℃30秒(30个循环)，72℃5分钟。常规方法提取质粒。用DraI(NEB,R0129)酶切后，胶回收约200bp的片段，用MEGAshortscript kit(Ambion,AM1354)体外转录成gRNA，并纯化。Cas9蛋白购自Life Technologies(B25640)。将Cas9蛋白与gRNA混合，并加入少量无RNA酶的酚红，终浓度为Cas9 100ng/μL及gRNA 30ng/μL。
3)显微注射小体鲟1细胞期受精卵
将步骤1)制备的受精时间不超过20分钟的小体鲟1细胞期受精卵放置在略覆盖有0.3×Da nieau buffer的玻璃培养皿中。在体式显微镜(Olympus,日本)下调整小体鲟胚胎动物极朝上。利用氮气加压的定量显微注射系统(Warner PLI-100A，美国)，将Cas9蛋白与gRNA的混合溶液逐枚注射到小体鲟1细胞期受精卵动物极的受精孔中，每枚受精卵注射体积为2nL。将显微注射后的胚胎置于16℃培养，存活率为93.9％。
4)筛选靶基因ntl突变的小体鲟胚胎
待受精后8天，显微注射的胚胎发育至出膜期，按个体收集胚胎并以常规方法提取基因组DNA。用检测引物F2-GGAGAGCGAATTTCAGAA和R2-GCGCAATGTCATTT TAATAC扩增包含靶位点的基因部分片段。PCR反应条件为：95℃1分钟，95℃15秒，52℃15秒，72℃30秒(30个循环)，72℃5分钟。最后将PCR产物置于95℃1分钟，然后缓慢降温至37℃。将最终的PCR产物以8％聚丙烯酰胺凝胶电泳(PAGE)，以80V电压电泳1.5小时，凝胶用EB染色。根据电泳条带数及亮度，按照文献[Chen J,Zhang X,Wang T,Li Z,Guan G,Hong Y(2012)Efficientdetection,qu antification and enrichment of subtle allelic alterations.DNARes 19:423-433]的方法计算靶位点发生突变的频率。根据检测结果，在7枚显微注射的胚胎中，均检测到靶位点的突变，最高突变率达83.1％(图3)，同时，发生较高频率突变的两枚胚胎呈现出脊柱弯曲、尾部变短的表型，这与斑马鱼ntl基因被敲降的表型类似。
实施例2：
选择不同鲟鱼的ntl基因作为靶基因，按照本领域的常规方式构建靶点gRNA，按照实施例1中的方法，在西伯利亚鲟(A.baerii)、施氏鲟(A.schrenckii)、俄罗斯鲟(A.gueldenstaedtii)中均成功进行了胚胎显微操作。
Claims (4)
1.一种鲟鱼CRISPR/Cas9基因编辑的方法，包括：将Cas9蛋白与靶基因gRNA混合后，利用显微注射的方式，注射到受精时间不超过20分钟的鲟鱼1细胞期受精卵的动物极的受精孔中；
所述的受精卵的制备方法包括：挑选性成熟的鲟鱼亲鱼，在室内养殖系统中暂养；距人工繁殖36小时前，注射LHRHa和DOM混合物进行人工催产，注射剂量为10μg LHRHa+1mg DOM/kg体重，每隔12个小时注射1次，雌性鲟鱼连续注射2次，雄性鲟鱼注射1次；待亲鱼有排精/排卵迹象，挤出精液到干燥的烧杯，挤出卵到玻璃平皿；用0.3×Daniea u buffer将精液稀释100倍，进行人工授精；受精时间1分钟，随后洗去多余的精液，即获得可用于显微注射的鲟鱼受精卵；
所述的Danieau buffer为：17mM NaCl，2mM KCl，0.12mM MgSO4，1.8mM Ca(NO3)2，1.5mMHEPES，pH 7.6。
2.根据权利要求1所述的方法，所述的鲟鱼包括小体鲟，西伯利亚鲟(A.baerii)、施氏鲟(A.schrenckii)、俄罗斯鲟(A.gueldenstaedtii)。
3.根据权利要求1所述的方法，所述的Cas9蛋白与靶基因gRNA的终浓度分别为100ng/μL和30ng/μL。
4.根据权利要求1所述的方法，每个受精卵注射体积为2nL。
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