CN108251452A - 一种表达Cas9基因的转基因斑马鱼及其构建方法和应用 - Google Patents
一种表达Cas9基因的转基因斑马鱼及其构建方法和应用 Download PDFInfo
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Abstract
本发明涉及一种表达Cas9基因的转基因斑马鱼及其构建方法和应用。所述的表达Cas9基因的转基因斑马鱼，是在斑马鱼Mitfα基因中插入了Cas9基因。本发明利用CRISPR/Cas9转基因技术在色素基因Mitfα定点靶向构建了可表达Cas9基因的转基因斑马鱼，并利用色素消退作为筛选纯合敲除品系的标志。通过使用该转基因斑马鱼对Tyr基因和ZFERV基因进行编辑，证实了该转基因斑马鱼在基因敲除中更加有效，并且在F0代即可获得纯合敲除个体。Cas9转基因斑马鱼与传统的CRISPR/Cas9技术在斑马鱼中的运用相比，具有更高的基因编辑效率；为大规模的基因筛选和疾病模型的建立提供了一个简单有效的工具。
Description
技术领域
本发明涉及转基因研究领域，具体涉及利用基因组编辑技术构建一种表达Cas9基因的转基因斑马鱼的方法。
背景技术
早在上世纪30年代，斑马鱼就成为生物医学研究的经典胚胎发育模型。从那时起，斑马就鱼首次被应用于研究脊椎动物的胚胎发育等相关问题。近十年来，ZFNs、TALENs和CRISPR/Cas9等基因编辑技术先后应用于鱼类基因功能研究中，为基因功能研究开辟了新途径。这些基于核酸的基因组编辑工具通过使用不同的识别模块靶向感兴趣的特定位点，在特定的基因位点精确诱导双链断裂，并通过非同源重组方式修复DNA。
这些基因编辑工具在斑马鱼研究中的使用具有划时代意义，为遗传学、发育生物学、毒理学、药物发现等提供了新的研究手段。然而利用传统方法获得纯合的敲除鱼难度较大，耗时费力。实际上，传统方法获得的斑马鱼胚胎(F0)大多为嵌合体，因此在F1后代中需要进行检测以确定突变的发生。此外，如果需要纯合突变，则F2后代必须对其进行分型鉴定，可以将携带相同突变的杂合F1鱼交配，以期在下一代(F2)中筛选到纯合突变体。
CRISPR/Cas9系统与ZFNs和TALENs相比，操作简单，成本较低，并且更易于大规模展开筛选，其已经被应用于各种细胞类型和生物体中。在CRISPR/Cas9系统的应用过程中发现，Cas9蛋白的功效已成为其基因编辑效率的制约因素。在比较注射混合物中的Cas9成分是mRNA或蛋白质对CRISPR系统效率的影响时发现，如果在某种程度上使Cas9蛋白效率恒定，CRISPR系统的基因编辑效率将获得巨大提高。
本发明就是为了解决上述现有技术存在的问题而构建了Cas9Mitfa-/-转基因斑马鱼品系(命名为：TU(ef1α：Cas9)Mitfα)。其中，Mitfa基因与斑马鱼黑色素沉积相关，利用色素消退表型利于筛选，且Mitfa基因缺陷对斑马鱼正常生理功能没有影响。这种转基因斑马鱼可以与多种sgRNA相搭配，为研究基因功能及其与相关疾病的相互作用提供了一个重要工具。
发明内容
本发明是利用日趋完善的CRISPR系统将Cas9基因定点导入斑马鱼基因组Mitfα色素基因位点，构建以色素消退为筛选特征的Cas9转基因斑马鱼。
本发明的原理是：在斑马鱼Mitfα基因上设计相应的sgRNA序列，利用CRISPR/Cas9系统的定点切割功能，并通过同源重组的方式将Cas9基因完整表达框插入该切割位点，通过观察色素消退的现象对纯合插入的转基因斑马鱼进行筛选并建立纯合敲除系。
本发明公开了一种表达Cas9基因的转基因斑马鱼，是在斑马鱼Mitfα基因中插入了Cas9基因。
本发明还提供了所述的表达Cas9基因的转基因斑马鱼在CRISPR/Cas9基因编辑及功能研究中的应用。
本发明所述的表达Cas9基因的转基因斑马鱼，是以色素消退作为转基因斑马鱼纯系建立筛选标志。
本发明还进一步提供了所述表达Cas9基因的转基因斑马鱼的构建方法：将Cas9基因定点插入到斑马鱼基因组的Mitfa基因中；在F1代及后续的子代筛选出色素消退的斑马鱼个体即可建立纯合Cas9Mitfa-/-斑马鱼品系。
本专利中通过将斑马鱼自身的Ef1α启动子结合到MLM3613载体中的Cas9基因前端，以该重组质粒为模板扩增出用于同源重组转基因操作的供体片段，将其利用CRISPR系统的定向切割功能，定点导入到斑马鱼基因组中Mitfα基因位点中，构建Cas9Mitfa-/-斑马鱼品系。由于选用的Mitfa基因与斑马鱼黑色素沉积相关，所以在F1代及后续的子代筛查中仅需要筛选出色素消退的斑马鱼个体即可建立纯合Cas9Mitfa-/-斑马鱼品系，同时Mitfa基因缺陷对斑马鱼正常生理功能没有影响。
现有的利用CRISPR系统进行斑马鱼基因功能研究方面虽然相较以往的工具(如ZFNs，TALENs及转座子系统等)更加便捷高效且具有靶向性，但是在后续的筛选过程中和以往的基因编辑系统一样存在需要大量人工和物力投入的情况。本发明利用CRISPR/Cas9转基因技术在色素基因Mitfα(ID:NM_130923)定点靶向构建了可表达Cas9基因的转基因斑马鱼，并利用色素消退作为筛选纯合敲除品系的标志。通过使用该转基因斑马鱼对Tyr基因(ID:NM_131013)和ZFERV(ID:AF503912)基因进行编辑，证实了该转基因斑马鱼在基因敲除中更加有效，并且在F0代即可获得纯合敲除个体。Cas9转基因斑马鱼与传统的CRISPR/Cas9技术在斑马鱼中的运用相比，具有更高的基因编辑效率。本发明利用色素消退这一现象作为筛选标志，极大的简化了建立纯系过程中的人力和物力消耗；同时，构建的表达Cas9基因的斑马鱼相比较原有的基于CRISPR系统的操作来说，在靶位点的切割效率上更胜一筹。由于使用斑马鱼自身的Ef1α启动子启动Cas9基因，可以在斑马鱼胚胎发育的很早期就表达Cas9蛋白，仅需注射sgRNA，就可对相应的剪切位点进行切割，相比原有的注射Cas9mRNA或者Cas9蛋白的情况，本方法构建的表达Cas9基因的斑马鱼不仅在单一位点的切割效率和持续性上具有优势，不会出现反应时间不充分，切割不完全的情况，同时在后续的子代转基因斑马鱼中Cas9基因的表达稳定。相比传统操作，嵌合子出现频率更低，并且由于鱼体色素消退，为后续的筛选和表达示踪提供了便捷。此工具对高效建立人类相关疾病模型，以及多基因功能研究均具有重要意义。
附图说明
图1：Cas9基因整合进入斑马鱼Mitfa基因示意图,同源臂序列HR-F、HR-R，斑马鱼自身启动子序列Ef1α-F、Ef1α-R；
图2：1到23是F0代斑马鱼Cas9基因检测结果，基因片段长453bp，M为DL2000Marker(TAKARA 3427A)，检测引物：Cas9-test-F、Cas9-test-R；
图3：转Cas9基因斑马鱼Mitfa基因位点插入检测结果，TU斑马鱼作对照，基因片段长1383bp，M是DL2000Marker(TAKARA 3427A)，检测引物：Cas9insertion-test-F、Cas9insertion-test-R；
图4：纯合Cas9Mitfa-/-斑马鱼品系构建示意图；
图5：转基因斑马鱼F1代Cas9基因表达鉴定，M是DL2000Marker(TAKARA 3427A)，G是F1代基因组，RT-是F1代RNA，RT+是F1代RNA逆转录所得cDNA，所得条带与F0代斑马鱼Cas9基因检测条带长度一致，检测引物Cas9-test-F、Cas9-test-R。
图6：转基因组和对照组各时间点(24h、96h、1month)斑马鱼的形态对比图。
图7：编辑Tyr基因示意图。
图8：Tyr基因编辑检测(T7E1)。M是DL500Marker(TAKARA 3427A)，1是野生组，2是传统方法注射组，3是转Cas9基因斑马鱼注射组，检测引物：Tyr-test-F、Tyr-test-R。
图9：利用表达Cas9基因的杂合子敲除ZFERV基因示意图；
图10：ZFERV 5'LTR PCR鉴定检测示意图，M是DL2000Marker(TAKARA 3427A)。1为24hpf的胚胎样本，2为48hpf脊柱畸形样本，3为120hpf脊柱畸形样本，4是TU对照组，左侧为斑马鱼内参基因GapDH检测，右侧为5'LTR检测；检测引物：ZB GapdH-F、ZB GapdH-R、5’LTR-test-F、5’LTR-test-R。
图11：ZFERV基因敲除鉴定检测示意图，WT是野生对照组，KO是ZFERV基因敲除组，M是DL2000Marker(TAKARA 3427A),检测引物5’LTR-test-F、3’LTR-test-R；
图12：ZFERV基因敲除鱼与野生型鱼的形态学变化对比图。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
1.sgRNA合成
以下实例中sgRNA的合成均依照如下步骤，选用Super-Fidelity DNAPolymerase(Vazyme)和T7in vitro Transcription kit(BioLabs)合成。
(1)sgRNA模板的制备
体系：5×Buffer5μL，
dNTP(10mM)0.5μL，
sgRNA-F(μM)1μL，
sgRNA-R(μM)1μL，
DNA Polymerase0.5μL，
RNase-free water 17μL，
Total25μL。
条件：98℃2min，50℃10min，72℃10min。
(2)产物回收
取2μL产物，用2.5％琼脂糖凝胶进行电泳检测。
(3)sgRNA体外转录
条件：37℃过夜。
(4)体外纯化(去除游离碱基、T7酶、模板DNA)
1)sgRNA体外转录产物加RNase-free水补足至100μL。加入等体积(各100μL)的Tris饱和酚-氯仿，15000rpm在4℃离心10min，缓慢吸取上清；
2)加入1mL 100％乙醇，震荡10秒后，15000rpm在4℃离心10分钟，弃上清；
3)加入1mL RNase-free 75％乙醇，震荡10秒后，15000rpm在4℃离心10分钟，弃上清；
4)晾干，所得沉淀即体外转录所得sgRNA。可溶于RNase-free水中后立即使用或放-80℃长期保存。
2.Cas9mRNA制备
(1)体外转录模板的制备
使用限制性内切酶PmeI线性化MLM3613-Cas9质粒，琼脂糖凝胶电泳确认线性化完全后，纯化得Cas9mRNA体外转录模板。
(2)Cas9mRNA的合成
选用mMESSAGEKit(Ambion)合成。步骤如下：
条件：37℃，1h。
(3)体外纯化(同上述1)
实施例1
本实施例1中，用斑马鱼Mitfα基因作为Cas9基因插入位点，利用显微注射的方法将Cas9mRNA、MitfasgRNA以及Cas9基因供体片段的混合液注射受精卵。鉴定斑马鱼F0代Cas9基因的整合情况，并检测其在子代(F1)中的表达，最终利用色素消退在F1代中挑选纯合子建系。
具体包括如下步骤：
(1)科学饲养斑马鱼，选取健康的雄鱼和雌鱼，通过自然交配(光照周期为：14h光照，10h黑暗,雌雄比2:1)产生受精卵，收集受精卵。
(2)用斑马鱼Mitfα基因作为Cas9基因整合位点，分别合成MitfαsgRNA和Cas9mRNA。将斑马鱼自身的Ef1α启动子结合到MLM3613载体中的Cas9基因前端，以该重组质粒为模板扩增出用于同源重组转基因操作的供体片段，Cas9基因后端依次连接NLS信号肽和bGH polyA，在Mitfα基因切割部位附近选择40bp长度的序列作为同源臂连接到供体序列两侧，利用CRISPR系统的定向切割功能，定点的导入到斑马鱼基因组中Mitfα基因位点中(图1)。
MitfαsgRNA及其引物序列：
Mitfa-sgRNA-F:TAATACGACTCACTATAGGCGCCGAGCACGGCATGACCCGTTTTAGAGCTAGAAATAGC(SEQ ID No.1)
Mitfa-sgRNA:CGCCGAGCACGGCATGACCC(SEQ ID No.2)
sgRNA-R:AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTGCTATTTCTAGCTCTAAAAC(SEQ ID No.3)
Ef1α启动子引物序列：Ef1α-F:CTGCAGATTTGGTAGATATCCATCAGTTCTAATG(SEQ IDNo.4)
Ef1α-R:GATTGATAAGTTTCTGCGGACCAAGATAAATGT(SEQ ID No.5)
同源臂序列：HR-F:TGAGCCCACAGGCCAGCACAGGGCCCGGCCCCAGCCAGCC(SEQ ID No.6)
HR-F:CGGGACCCGGAGCCAGCGCTCCCAACAGCCCTATGGCCCT(SEQ ID No.7)
(3)斑马鱼显微注射：5μL注射混合液含有Cas9mRNA1000ng、sgRNA 200ng和Cas9基因供体片段500ng，注射体积为每个胚胎1nL。注射后的胚胎置于温度为28℃的E3培养液中培养，并留少量胚胎不注射，留作TU对照组。同时观察24hpf、96hpf和1mpf时期TU斑马鱼形态，用作与纯合Cas9Mitfa-/-斑马鱼品系作同期对照。
(4)取1月龄的F0斑马鱼，挑选色素消退明显的斑马鱼个体，取部分尾鳍，提取基因组DNA鉴定F0代斑马鱼Cas9基因整合情况，并设TU对照提取基因组。Cas9基因检测PCR出现453bp长度条带，表明目的基因已整合到F0斑马鱼的基因组中(图2)。将已整合Cas9基因的个体基因组样本挑出，继续进行Mitfa位点插入鉴定。Mitfa位点插入检测PCR结果显示，扩增条带为1383bp，而TU组无条带扩增出来，表明Cas9基因片段已整合到斑马鱼Mitfα基因中(图3)。
基因组整合Cas9基因检测引物序列：
Cas9-test-F:CAAACGGACAGCTCGTAGAA(SEQ ID No.8)
Cas9-test-R:CGTGCAGAAAGAATCGCTTTAG(SEQ ID No.9)
Mitfa位点插入Cas9基因检测引物序列：
Cas9insertion-test-F:AAACAGAAATTACACTTGCAAATGGAGAG(SEQ ID No.10)Cas9insertion-test-R:CTCTTTCTCACAGTTGAGGGTG(SEQ ID No.11)
(5)将已定点整合Cas9基因的F1代转基因斑马鱼作为亲本，进行交配繁殖，跟据色素消退筛选出表达Cas9基因的纯合转基因斑马鱼F2代(图4)。对纯合转基因斑马鱼F1代进行基因组整合检测以及表达检测，检测结果显示Cas9基因已整合入基因组中且成功表达(图5)。转基因组和对照组各时间点(24hpf、96hpf、1mpf)斑马鱼的形态有所差异，可观察到表达Cas9基因的斑马鱼色素消退明显，体表无黑色素沉积(图6)。至此，纯合Cas9Mitfa-/-斑马鱼品系成功建立。(基因组整合Cas9基因检测引物Cas9-test同上)
实施例2
本实施例2中，通过对Tyr基因进行编辑，证明所构建的表达Cas9基因斑马鱼可以作为基因编辑工具。用转Cas9基因斑马鱼和野生型TU斑马鱼的胚胎作显微注射，并设置对照组。收集发育48hpf注射组样品，提取基因组DNA进行鉴定。
具体包括如下步骤：
(1)科学饲养斑马鱼，分别选择健康的野生斑马鱼和Cas9转基因斑马鱼的雄鱼和雌鱼，各自自然交配繁殖产生受精卵(光照周期为：14h光照，10h黑暗,雌雄比2:1)，并分别收集受精卵。设对照组(未注射)，传统方法胚胎注射组，Cas9转基因斑马鱼注射组。此外留部分转基因斑马鱼胚胎不作任何处理。
(2)用表达Cas9基因斑马鱼和野生型TU斑马鱼的受精卵作显微注射，选择斑马鱼Tyr基因，利用试剂盒合成Tyr sgRNA和Cas9mRNA。传统方法胚胎注射组混合液5μL含有Cas9mRNA1000ng和sgRNA 200ng表达Cas9基因斑马鱼胚胎注射组注射液5μL含有sgRNA200ng。注射体积为每个胚胎1nL，将胚胎置于温度为28℃的E3培养基中孵育。Tyr基因编辑示意图如图7。
TyrsgRNA及其引物序列：
Tyr-sgRNA-F:TAATACGACTCACTATAGGGCTGAGGAACCAATCAGCTGGTTTTAGAGCTAGAAATAGC(SEQ ID No.12)
Tyr-sgRNA:GCTGAGGAACCAATCAGCTG(SEQ ID No.13)
sgRNA-R:AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTGCTATTTCTAGCTCTAAAAC(SEQ ID No.14)
(3)分别收集培养48h的斑马鱼胚胎，提取基因组DNA，进行Tyr位点编辑检测。T7E1酶检测结果显示，TU组无突变型带，Cas9转基因斑马鱼注射组和传统注射组均出现突变型带，且前者突变条带更为明显(图8)。因此证实胚胎中Tyr基因位点被剪切且编辑效率高于传统注射方法，构建的表达Cas9基因的斑马鱼用于基因编辑是有效的。
Tyr位点编辑检测引物序列：
Tyr-test-F:AACATATGTGACCCGCATCA(SEQ ID No.15)
Tyr-test-R:TCATATTCTACTGTAATGTGAGTTTGA(SEQ ID No.16)
实施例3
本实施例3中，通过敲除斑马鱼内源性反转录病毒ZFERV基因，来检测Cas9转基因斑马鱼对基因组长片段编辑的效率。收集不同发育阶段的注射斑马鱼胚胎，提基因组DNA进行鉴定。检测发现，表型有显著差异的斑马鱼胚胎的ZFERV基因被完全敲除。
具体包括如下步骤：
(1)科学饲养斑马鱼，选择健康的野生TU斑马鱼自然交配产卵作为对照组，Cas9转基因斑马鱼与野生TU斑马鱼杂交产卵，并分别收集受精卵。
(2)针对ZFERV 5’LTR和3’LTR同源区域设计sgRNA，对表达Cas9基因的受精卵进行注射(图9)。
LTRsgRNA及其引物序列：
LTR-sgRNA-F:TAATACGACTCACTATAGGGCGTATCTCAGTCTGTGTAGGTTTTAGAGCTAGAAATAGC(SEQ ID No.17)
LTR-sgRNA:GCGTATCTCAGTCTGTGTAG(SEQ ID No.18)
sgRNA-R:AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTGCTATTTCTAGCTCTAAAAC(SEQ ID No.19)
(3)在不同发育阶段(24hpf，48hpf，120hpf)分别收集敲除组和对照组斑马鱼胚胎进行基因敲除鉴定，选用斑马鱼GapdH基因(ID:NM_001115114)作为内参。其中24hpf时期的斑马鱼胚胎还未出膜，无法分辨脊柱发育是否异常，48hpf之后脊柱长成，方可挑出脊柱发育异常的鱼苗，48hpf和120hpf时期挑选有显著表型差异的个体。通过5’LTR检测显示，24hpf时期和TU对照组均有266bp长度条带扩增出，表明24hpf时期挑选的样品为混合样；48hpf和120hpf时期样本则相对单一，无条带扩增出，因此可以确定ZFERV基因被完全敲除(图10)。通过使用ZFERV两端引物，在实验组中扩增出了一个短片段，测序结果显示为ZFERV敲除后剩余基因拼接产物，进一步验证ZFERV基因被完全敲除(图11)。胚胎发育一定时期后，敲除组脊柱发育异常，和对照组斑马鱼表形出现显著差异(图12)。综上可见，Cas9转基因斑马鱼对基因组长片段的编辑是有效的。
内参ZB-GapdH引物序列：
ZB GapdH-F:CATGTTCCAGTACGACTCCAC(SEQ ID No.20)
ZB GapdH-R:CATCAATGACCAGTTTGCCG(SEQ ID No.21)
5’LTR检测引物序列：
5’LTR-test-F:ATTGCTTTGTTTGAAATGTGAG(SEQ ID No.22)
5’LTR-test-R:CTCCAATCCATTATTTCGCTTC(SEQ ID No.23)
ZFERV两端检测引物：
5’LTR-test-F:ATTGCTTTGTTTGAAATGTGAG(SEQ ID No.24)
3’LTR-test-R:CAATATCTGCTTATAGTCCTCCTTTAACT(SEQ ID No.25)。
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Claims (2)
1.表达Cas9基因的转基因斑马鱼在CRISPR/Cas9基因编辑及功能研究中的应用。
2.一种表达Cas9基因的转基因斑马鱼的构建方法，其特征在于包括以下步骤：将Cas9基因定点插入到斑马鱼基因组的Mitfa基因中；在F1代及后续的子代筛选出色素消退的斑马鱼个体即可建立纯合Cas9 Mitfa-/-斑马鱼品系。
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