CN108148873A - 一种cav-1基因缺失斑马鱼及其制备方法 - Google Patents
一种cav-1基因缺失斑马鱼及其制备方法 Download PDFInfo
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Abstract
本发明提供一种CAV‑1基因缺失斑马鱼突变体及其制备方法，CAV‑1基因缺失斑马鱼突变体的构建是通过CRISPR/Cas9技术实现的。本发明的突变体模型可用于研究CAV‑1基因在肿瘤发生和远端转移过程中的作用。
Description
技术领域
本发明涉及分子生物学领域，涉及一种基因敲除的斑马鱼突变体，具体涉及CAV-1基因缺失斑马鱼突变体及其制备方法。
背景技术
成簇规律间隔回文重复序列CRISPR(Clustered Regularly Interspaced ShortPalind-romic Repeats)和CRISPR相关核酸酶Cas(CRISPR-associated nuclease)系统是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御机制。该系统能够识别自身序列和外源入侵DNA 片段，并剪切掉外源片段，从而达到保护自己的目的。科学家将这种免疫机制开发成为一种基因定点编辑技术，广泛应用于生物学和医学研究，目前最常见的基因编辑技术为 CRISPR/Cas9系统。该系统通过向导RNA(single guide RNA,sgRNA)序列，一方面特异识别靶序列，另一方面引导Cas9蛋白定点切割靶位点，使DNA靶位点形成双链缺口，细胞通过同源重组修复或者非同源性末端连接对断裂的DNA进行修复，从而实现基因的编辑。
Caveolae结构细胞质膜向内凹陷所形成的囊泡状结构，参与细胞内外物质转运和细胞信号传导过程。微囊蛋白-1(Caveolin-1)是Caveolae结构的主要成分，对许多关键信号分子的活性状态起直接调节作用。有研究表明，Caveolin-1与肿瘤的发生与远处转移、癌细胞的转化等过程相关。CAV-1基因是编码Caveolin-1蛋白的DNA序列，可以通过对CAV-1基因进行定点编辑，从而研究Caveolin-1蛋白在肿瘤中的具体作用，以实现治疗疾病的目的。
斑马鱼具有生长发育快，繁殖周期短、子代数量多、体外受精、胚胎透明等生物学特性，十分适合进行基因编辑。构建CAV-1基因突变斑马鱼作为疾病模型，能深入探究CAV-1基因与相关疾病的联系及发生机制，为靶向药物的筛选提供一种稳定可靠的模式生物。
发明内容
基于此，本发明的目的在于，提供一种CAV-1基因缺失斑马鱼及其制备方法。
为了实现上述目的，本发明采用了如下技术方案：
一种CAV-1基因敲除方法，其过程包括，设计识别CAV-1基因靶位点的sgRNA序列，所述的sgRNA序列与核酸酶结合并引导核酸酶结合到CAV-1基因靶位点处，核酸酶对靶位点处的序列进行随机剪切，通过细胞自身的非同源末端连接修复机制修复CAV-1基因双链，造成移码突变，完成CAV-1基因被敲除。
进一步地，所述的sgRNA序列具有序列表中SEQ ID NO:1或SEQ ID NO:2所示的核苷酸序列；所述的核酸酶为Cas9蛋白。
进一步地，所述的SEQ ID NO:1记载的核苷酸序列含有HaeⅡ酶切位点；所述的SEQID NO:2记载的核苷酸序列含有StyⅠ酶切位点。
本发明所述的CAV-1基因缺失斑马鱼突变体的制备方法，包括以下步骤：
1)设计识别CAV-1基因靶位点的sgRNA序列，所述的sgRNA序列具有序列表中SEQID NO:1或SEQ ID NO:2所示的核苷酸序列；
2)将所述的sgRNA序列和Cas9mRNA共同显微注射到斑马鱼胚胎中，然后对得到的斑马鱼胚胎进行培养，得到所述的CAV-1基因缺失斑马鱼突变体。
进一步地，所述步骤1)具体包括：查询斑马鱼CAV-1基因序列及功能结构域，结合CRISPR/Cas9敲除原理，设计并合成包含sgRNA序列的引物序列，然后通过PCR扩增得到大量双链sgRNA序列，接着通过体外转录双链sgRNA序列得到单链sgRNA序列。
进一步地，所述的引物序列具有序列表中SEQ ID NO:3或者SEQ ID NO:4所示的核苷酸序列，以及具有序列表中SEQ ID NO:5所示的核苷酸序列。
进一步地，步骤2)中，所述的Cas9mRNA来自于pGH-T7-zCas9质粒，XbaI酶切 pGH-T7-zCas9质粒得到Cas9DNA双链，然后通过体外转录Cas9DNA双链得到Cas9mRNA单链。
进一步地，步骤2)中，所述的斑马鱼胚胎为单细胞期胚胎，显微注射剂量为2nL；其中， sgRNA的注射浓度为300ng/μL，Cas9mRNA的注射浓度为120ng/μL。
一种CAV-1基因缺失斑马鱼突变体的检测方法，包括以下步骤：以待检测的斑马鱼基因组DNA为模板，PCR扩增含有权利要求2中所述的sgRNA序列的DNA片段，然后HaeⅡ或者StyⅠ酶切该DNA片段，实现CAV-1基因缺失斑马鱼突变体的检测。
进一步地，所述PCR扩增的引物序列具有序列表中SEQ ID NO:6和SEQ ID NO:7所示的核苷酸序列，或者具有序列表中SEQ ID NO:8和SEQ ID NO:9所示的核苷酸序列。
本发明基于CAV-1基因与肿瘤的发生和远端转移、癌细胞的转化有关，构建了CAV-1基因缺失斑马鱼突变体，方便对CAV-1基因在疾病中的作用进行在体研究。
附图说明
图1为CAV-1突变体的繁育及筛选流程图；
图2为XbaI酶切pGH-T7-zCas9质粒示意图；
图3为显微注射和Cas9蛋白工作原理示意图；
图4为显微注射胚胎CAV-1靶位点1酶切鉴定电泳图；
图5为显微注射胚胎CAV-1靶位点2酶切鉴定电泳图；
图6为F1的CAV-1靶位点1酶切鉴定电泳图；
图7为F1的CAV-1靶位点2酶切鉴定电泳图；
图8为qPCR检测F2突变体的CAV-1的mRNA表达水平柱状图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例一：选择斑马鱼CAV-1基因靶位点
查找斑马鱼CAV-1基因生物信息，基于CAV-1基因编码的蛋白Caveolin-1具有跨膜物质运输和信号传导作用，因此，将靶位点设置在CAV-1基因编码跨膜域的位置。同时，为了便于检测，靶位点序列中最好含有酶切位点。
靶位点1位于Caveolin-1蛋白跨膜域5’端交界处，该序列中含有HaeⅡ酶切位点；靶位点2位于Caveolin-1蛋白跨膜域5’端前159碱基处，该序列中含有StyⅠ酶切位点。靶位点的核苷酸序列如下：
靶位点1：ggctgctgacagcgctggt；(下划线部分为HaeⅡ酶切位点)
靶位点2：gaccaggtcaatctccttggtg。(下划线部分为StyⅠ酶切位点)
实施例二：筛选斑马鱼亲本
单核苷酸多态性(Single nucleotide polymorphism,SNP)是指在基因组水平上由单个核苷酸的变异而引起的DNA序列多态性，是自然界中的普遍现象。为了使sgRNA序列精确识别靶位点，需要筛选靶位点序列不存在SNP的胚胎，即需要筛选靶位点不存在SNP的斑马鱼亲本。
本发明通过扩增包含靶位点序列的片段，对其测序，并比对其与靶位点序列的一致性，进行斑马鱼亲本的筛选。具体过程如下：
选取6～8对AB品系野生型斑马鱼，剪背鳍，抽RNA并反转录得到cDNA，该cDNA作为PCR扩增模板。根据CAV-1基因靶位点处的序列，设计特异性引物，使得扩增得到的序列包含完整的靶位点序列，且只含有一个HaeⅡ或者StyⅠ酶切位点。引物如下：
靶位点1的正向引物：aggtcttagaatccagtcagtg；反向引物：cactctgtccttacaccacc。
靶位点2的正向引物：gtgtggtgcttcatttctatcc；反向引物：atggtcttacttagcagtgtg。
包含靶位点1序列在内的片段长度为472bp，只含有一个HaeⅡ酶切位点；包含靶位点2 序列在内的片段长度为574bp，只含有一个StyⅠ酶切位点。
PCR反应体系如下(25μL)：
PCR反应条件如下：94℃预变性5分钟，94℃变性30秒，59℃退火30秒，72℃延伸30秒， 40个循环，最后72℃延伸7分钟。其中，包含靶点1序列片段的PCR退火温度为59℃，而包含靶点2序列片段的PCR退火温度为56℃。
对所得的PCR产物进行测序，并将测序结果与靶位点序列进行比对，保留与靶位点序列完全一致的成鱼，即为靶位点无SNP的斑马鱼成鱼。以靶位点无SNP的斑马鱼成鱼为亲本，交配获得靶位点无SNP的单细胞胚胎，该胚胎用于显微注射。
实施例三：体外合成sgRNA
本发明采用T7转录体系进行sgRNA的转录。sgRNA序列能够引导Cas9蛋白剪切靶位点处的DNA序列，其序列包含两部分：一部分与Cas9蛋白结合；另一部分识别靶位点序列。根据 CAV-1基因靶位点序列，设计T7启动子的下游区域，使得最终转录出来的RNA序列包含sgRNA的完整序列。
根据T7启动子序列和CAV-1基因靶位点序列，设计特异性引物，如下：
靶位点1的正向引物：taatacgactcactatagggctgctgacagcgctggtgttttagagctagaaatagc；(斜体部分是T7启动子序列，下划线部分是靶位点1序列)
靶位点1的反向引物：agcaccgactcggtgccact。
靶位点2的正向引物：taatacgactcactataggaccaggtcaatctccttggtggttttagagctagaaatagc；(斜体部分是T7启动子序列，下划线部分是靶位点2序列)
靶位点2的反向引物：agcaccgactcggtgccact。
以sgRNA的骨架载体pT7-gRNA质粒为模板，和上述引物进行PCR扩增。所得扩增产物作为体外转录模板。该转录模板序列按功能可分为三个区域：分别是转录启动子区、sgRNA 基因靶向序列和Cas9蛋白铆定序列。
PCR反应体系如下(50.0μL)：
PCR反应条件如下：94℃预变性5分钟，94℃变性30秒，60℃退火30秒，72℃延伸30秒，40个循环，最后72℃延伸7分钟。
所得的PCR产物经纯化处理后，作为体外转录sgRNA的模板，用T7聚合酶介导的转录反应体系进行体外转录。
体外转录反应体系如下(50.0μL)：
体外转录反应条件如下：37℃下转录反应3小时。
体外转录所得的sgRNA中含有DNA，通过加入DNase1去除DNA只保留sgRNA，反应体系如下：
sgRNA 50.0μL，
DNase1 2.0μL。
反应条件如下：37℃反应15～30分钟。
体外转录得到的sgRNA经去DNA和纯化后，通过琼脂糖凝胶电泳进行分析，并用超微量紫外分光光度计进行定量，随后于-80℃储存。
实施例四：制备Cas9mRNA
本发明的Cas9基因序列来自于pGH-T7-zCas9质粒。如图2所示，pGH-T7-zCas9质粒经XbaⅠ酶切后，可得到含有Amp基因序列、T7启动子序列、核定位信号(Nuclear SignalLocalization，NSL)序列、Cas9基因序列和核定位信号系列的片段。
酶切反应体系如下(50.0μL)：
酶切反应条件如下：37℃下酶切反应10小时。
所得的酶切产物经纯化处理后，作为体外转录Cas9mRNA的模板，用T7聚合酶介导的转录反应体系进行体外转录。
体外转录反应体系如下(20.0μL)：
体外转录反应条件如下：37℃下转录反应2小时。
体外转录所得的Cas9mRNA中含有DNA，通过加入DNase1去除DNA只保留Cas9 mRNA，反应体系如下：
Cas9mRNA 20.0μL，
DNase1 2.0μL。
反应条件如下：37℃反应15～30分钟。
去除DNA之后的Cas9mRNA经氯化锂沉淀法回收并纯化后，通过聚丙烯酰胺凝胶电泳 (PAGE)进行分析，并用超微量紫外分光光度计进行定量，随后于-80℃储存。
实施例五：显微注射斑马鱼胚胎
选择靶位点无SNP的雌雄鱼交配得到受精卵，约18h后取上述受精卵进行显微注射。显微注射前，先混匀sgRNA和Cas9mRNA，其中，sgRNA的终浓度为300ng/μL，Cas9mRNA 的终浓度为120ng/μL。
如图3所示，在显微镜下，将预混好的Cas9mRNA和sgRNA溶液注射入单细胞期斑马鱼胚胎中，每个受精卵注射剂量为2nL。至少注射200枚胚胎，同时预留部分未经注射的同批胚胎作为实验对照。注射胚胎2小时后，于显微镜下挑出未受精和已死亡的胚胎，正常胚胎放入孵育溶液中培养。
胚胎孵育溶液的配方如下(800mL)：
水 800mL，
美兰溶液 1～2滴。
胚胎孵育条件如下：28.5℃条件下培养至受精后5天，在此期间每12小时更换孵育溶液，同时清除死卵并记录死亡情况。
实施例六：Cas9突变效率检测
从注射组中随机挑取5～10条受精后一天的(1day post fertilization，1dpf)无畸形的胚胎，提取胚胎基因组DNA。以该基因组DNA为模板，与序列表中的SEQ ID NO:6和SEQID NO:7所示的核苷酸序列为引物，或者与序列表中的SEQ ID NO:8和SEQ ID NO:9所示的核苷酸序列为引物进行PCR扩增。PCR反应体系与条件参照实施例1。由于该PCR产物包含靶位点序列，和唯一的HaeⅡ或Sty I酶切位点，因此，对该片段进行酶切检测即可掌握靶位点突变情况。
为了确定酶切是否完全，实验需选取同一批次未经注射的胚胎作为对照组。
酶切体系如下：(10.0μL)
酶切反应条件如下：37℃下酶切反应10～12h。其中，PCR产物为含有靶位点1序列的片段时，使用HaeⅡ酶，PCR产物为含有靶位点2序列的片段时，使用StyⅠ酶。
CAV-1基因靶位点1突变效率检测结果如图4所示。经过PCR扩增，实验组(第3～24条带)和对照组(第1～2条带)均得到471bp长度的片段。该片段含有靶位点1序列，即含有一个 HaeⅡ酶切位点。
上述片段经过HaeⅡ酶切反应后，阳性对照组，即加入HaeⅡ的第1条带被完全切开；阴性对照组，未加入HaeⅡ的第2条带未被切开；实验组(第3～24条带)中，除了第20条带被HaeⅡ完全切开之外，其余21条条带均未被完全切开，且其最上面的条带与阴性对照组位置相同。未能被HaeⅡ酶完全切开，说明该酶切位点的序列已被破坏，HaeⅡ限制性内切酶无法识别破坏后的酶切位点，故而不能够完全酶切片段。
本实验结果表明：经过注射包含靶位点1序列的sgRNA和Cas9mRNA的22个斑马鱼胚胎，有21个胚胎的靶位点1序列已被敲除，存在潜在突变，并且本次靶位点1的突变效率为21条:22条＝95.5％。
CAV-1基因靶位点1突变效率检测结果如图5所示。经过PCR扩增，实验组(第3～16条带)和对照组(第1～2条带)均得到573bp长度的片段。该片段含有靶位点2序列，即含有一个 StyⅠ酶切位点。
上述片段经过StyⅠ酶切之后，阳性对照组，即加入StyⅠ的第1条带被完全切开；阴性对照组，未加入StyⅠ的第2条带未被切开；实验组(第3～16条带)中，第3、4、6、8、9、 10、11、12、13条带未被StyⅠ完全切开之外，且其最上面的条带与阴性对照组位置相同。未能被StyⅠ酶完全切开，说明该酶切位点的序列已被破坏，StyⅠ限制性内切酶无法识别破坏后的酶切位点，故而不能够完全酶切片段。
本实验结果表明：经过注射包含靶位点2序列的sgRNA和Cas9mRNA的14个斑马鱼胚胎，有9个胚胎的靶位点2序列已被敲除，存在潜在突变，且本次靶位点2的突变效率为9 条:14条＝64.3％。
实施例七：筛选突变的成鱼
饲养注射后未畸形的胚胎至3月龄，剪其尾鳍，筛选具有潜在突变的F0。逐条剪尾鳍并分别提基因组DNA，以该基因组DNA为模板，与实施例1中的引物进行PCR扩增并酶切该PCR片段。通过PCR扩增含靶点序列和酶切检测靶位点的突变情况逐条进行检测，筛选出有潜在突变F0。选取具有潜在突变的F0与无SNP的AB品系斑马鱼杂交，得到F1胚胎，饲养胚胎至适合剪尾鳍。同理筛选F1突变体。F1突变体与健康成熟已检测无SNP的野生型AB 杂交，大量繁殖F2。
斑马鱼饲养条件如下：pH 7.2～7.6，水温28.5～29.5℃，每日光照时间14小时，8:30～22:30。另外，根据食物和环境的变化，将斑马鱼的饲养分为两个阶段。第一阶段，将受精后5天(5 days post fertilization，5dpf)的胚胎从28.5℃恒温箱转置于小缸中饲养至受精后15天(15days post fertilization，15dpf)，在此期间，每日喂食2～3次虾虫，同时每日更换养殖水；第二阶段，将15天的幼鱼转置于循环开放的大缸中饲养至3月龄，受精后15天至30天期间，每日两次喂食虾虫，受精后30天(30days post fertilization，30dpf)开始逐渐添加丰年虾，2月龄开始每日上午喂食粗虾粉，下午喂食丰年虾，期间每周定时清洗鱼缸。
实施例八：检测突变体基因型
经过筛选的F1突变体，以其基因组DNA为模板，PCR扩增含有靶位点序列的片段，并对该片段进行测序。将测序结果与原序列进行比对，从而确定突变体的基因型。实验结果显示，靶位点1的突变基因型有10种，分为两类。第一类基因型：缺失或增加的碱基数量为3 的倍数，包括(-3+9)、(-4+10)、(-6)、(-12)、(-15)5种基因型；第二类基因型：缺失或增加的碱基数量非3的倍数，包括(-13)、(-6+31)、(-8)、(-25+3)、(-17+4) 5种基因型。
实施例九：检测突变的遗传效率
选取有潜在突变的F0突变体与无SNP的AB品系斑马鱼杂交，获取F1。随机挑取10个以上F1胚胎，分别提取基因组DNA。PCR扩增含有靶位点序列的片段，并酶切该片段，检测F1靶位点的突变情况，从而获得F0的突变遗传效率。
随机挑取22条F1胚胎作为实验组(第1～22条带)，和2条AB品系胚胎(第23～24 条带)作为对照组。分别提取基因组DNA并PCR扩增含靶位点1的片段，电泳跑胶结果，如图6所示，均可见约471bp长度的条带。经HaeⅡ酶切后，对照组条带均被完全切开，实验组有16条未被完全切开。本次实验结果表明：CAV-1基因靶位点1突变体的F0遗传效率为16条:22条＝72.7％。
同理随机挑取12条F1胚胎实验组(第1～12条带)，取2条AB品系胚胎(第13～14条带)作为对照组。分别提取基因组DNA并PCR扩增含靶位点2的片段，电泳跑胶结果，如图7所示，均可见573bp长度的条带。经StyI酶切后，对照组条带均被完全切开，实验组有 2条未被完全切开。本次实验结果表明：CAV-1基因靶位点2突变体的F0遗传效率为2条:12 条＝16.7％。
实施例十：检测F2中CAV-1mRNA表达水平
将CAV-1基因靶位点1的10种F1基因型突变体培养为成鱼，与无SNP的AB品系斑马鱼交配，获得F2。提取F2个体的RNA，通过反转录得到cDNA。以该cDNA为模板，进行实时荧光定量核酸扩增(Real-time Quantitative PCR，qPCR)，检测F2个体的CAV-1mRNA 表达水平。qPCR实验以RPP0为内参基因，设计RPP0和CAV-1的特异性引物如下：
内参RPP0基因的正向引物：ctgaacatctcgcccttctc；
RPP0基因的反向引物：tagccgatctgcagacacac。
CAV-1基因的正向引物：ggaaccgcaggaatacgct；
CAV-1基因的正向引物：ctgaagtgtcttttcgttgatgct。
qPCR反应体系如下：(10.0μL)
qPCR反应条件如下：95℃预变性10分钟，95℃变性10秒，60℃退火10秒，72℃延伸10秒，45个循环，最后95℃反应10秒，65℃反应60秒，97℃反应1秒。
本实验结果，如图8所示，相对于无SNP的AB品系斑马鱼，F2个体中CAV-1的mRNA 水平明显较低，尤其是基因型为(-25+3)和(-6+31)的突变体，CAV-1的mRNA水平更低。本实验结果表明：CAV-1基因被有效敲除，且该敲除可遗传，缺失或增加碱基数量为非3倍数的基因型敲除效果更佳。
序列表
<110> 南方医科大学
<120> 一种CAV-1基因缺失斑马鱼及其制备方法
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggctgctgac agcgctggt 19
<210> 2
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaccaggtca atctccttgg tg 22
<210> 3
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
taatacgact cactataggg ctgctgacag cgctggtgtt ttagagctag aaatagc 57
<210> 4
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
taatacgact cactatagga ccaggtcaat ctccttggtg gttttagagc tagaaatagc 60
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agcaccgact cggtgccact 20
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aggtcttaga atccagtcag tg 22
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cactctgtcc ttacaccacc 20
<210> 8
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gtgtggtgct tcatttctat cc 22
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<213> 人工序列(Artificial Sequence)
<400> 9
atggtcttac ttagcagtgt g 21
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctgaacatct cgcccttctc 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tagccgatct gcagacacac 20
<210> 12
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggaaccgcag gaatacgct 19
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ctgaagtgtc ttttcgttga tgct 24
Claims (10)
1.一种CAV-1基因敲除方法，其特征在于，设计识别CAV-1基因靶位点的sgRNA序列，所述的sgRNA序列与核酸酶结合并引导核酸酶结合到CAV-1基因靶位点处，核酸酶对靶位点处的序列进行随机剪切，通过细胞自身的非同源末端连接修复机制修复CAV-1基因双链，造成移码突变，完成CAV-1基因被敲除。
2.根据权利要求1所述的CAV-1基因敲除方法，其特征在于，所述的sgRNA序列具有序列表中SEQ ID NO:1或SEQ ID NO:2所示的核苷酸序列；所述的核酸酶为Cas9蛋白。
3.根据权利要求2所述的CAV-1基因敲除方法，其特征在于，所述的SEQ ID NO:1记载的核苷酸序列含有HaeⅡ酶切位点；所述的SEQ ID NO:2记载的核苷酸序列含有StyⅠ酶切位点。
4.一种CAV-1基因缺失斑马鱼突变体的制备方法，其特征在于，所述的制备方法包括以下步骤：
1)设计并合成识别CAV-1基因靶位点的sgRNA序列，所述的sgRNA序列具有序列表中SEQID NO:1或SEQ ID NO:2所示的核苷酸序列；
2)将所述的sgRNA序列和Cas9mRNA共同显微注射到斑马鱼胚胎中，然后对得到的斑马鱼胚胎进行培养，得到所述的CAV-1基因缺失斑马鱼突变体。
5.根据权利要求4所述的CAV-1基因缺失斑马鱼突变体的制备方法，其特征在于，所述步骤1)具体包括：查询斑马鱼CAV-1基因序列及功能结构域，结合CRISPR/Cas9敲除原理，设计并合成包含sgRNA序列的引物序列，然后通过PCR扩增得到大量双链sgRNA序列，接着通过体外转录双链sgRNA序列得到单链sgRNA序列。
6.根据权利要求5所述的CAV-1基因缺失斑马鱼突变体的制备方法，其特征在于，所述的引物序列具有序列表中SEQ ID NO:3或者SEQ ID NO:4所示的核苷酸序列，以及具有序列表中SEQ ID NO:5所示的核苷酸序列。
7.根据权利要求4所述的CAV-1基因缺失斑马鱼突变体的制备方法，其特征在于，步骤2)中，所述的Cas9mRNA来自于pGH-T7-zCas9质粒，XbaⅠ酶切pGH-T7-zCas9质粒得到Cas9DNA双链，然后通过体外转录Cas9DNA双链得到Cas9mRNA单链。
8.根据权利要求4所述的CAV-1基因缺失斑马鱼突变体的制备方法，其特征在于，步骤2)中，所述的斑马鱼胚胎为单细胞期胚胎，显微注射剂量为2nL；其中，sgRNA的注射浓度为300ng/μL，Cas9mRNA的注射浓度为120ng/μL。
9.一种CAV-1基因缺失斑马鱼突变体的检测方法，其特征在于，包括以下步骤：以待检测的斑马鱼基因组DNA为模板，PCR扩增含有权利要求2中所述的sgRNA序列的DNA片段，然后HaeⅡ或者StyⅠ酶切该DNA片段，实现CAV-1基因缺失斑马鱼突变体的检测。
10.根据权利要求9所述的CAV-1基因缺失斑马鱼突变体的检测方法，其特征在于：所述PCR扩增的引物序列具有序列表中SEQ ID NO:6和SEQ ID NO:7所示的核苷酸序列，或者具有序列表中SEQ ID NO:8和SEQ ID NO:9所示的核苷酸序列。
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