CN106086061A - 一种基于CRISPR‑Cas9系统的酿酒酵母基因组编辑载体及其应用 - Google Patents
一种基于CRISPR‑Cas9系统的酿酒酵母基因组编辑载体及其应用 Download PDFInfo
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Abstract
本发明涉及一种基于CRISPR‑Cas9系统的酿酒酵母基因组编辑载体及其应用，所述的酿酒酵母基因组编辑载体通过将Cas9蛋白表达框和sgRNA scaffold表达框整合在载体中，获得载体‑Cas9‑sgRNA scaffold，然后将以ADE1为靶位点设计合成的多条sgRNA片段中的任意一条整合到所述的载体‑Cas9‑sgRNA scaffold中得到所述的酿酒酵母基因组编辑载体。本发明采用单质粒并单拷贝的载体系统，采用一个质粒同时表达Cas9蛋白和sgRNA scaffold，使得靶基因编辑系统只需一步构件和转化，操作简便。
Description
技术领域
本发明涉及一种基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体及其应用。
背景技术
随着人类全基因组测序的完成，生命科学研究进入了一个以揭示基因功能为目的的后基因组时代，基因组编辑技术成为了重要的研究工具和手段。传统的基因组编辑技术利用同源重组机制(homologous recombination)对基因进行定向编辑，可以帮助科研人员明确基因的功能，传统的基因组编辑技术是以长片段DNA为靶位点，“定位系统”也必须是长DNA片段，编辑系统构建复杂且费时，实验周期长，效率很低、且基因易突变等缺陷。第二代基因组编辑技术(包括ZFN系统和TALEN系统)在一定程度上解决了编辑系统特异性很低的问题，但是，依旧没能解决编辑系统构建繁琐，费时费力的突出问题。第三代基因组编辑技术CRISPR-Cas9基因组编辑系统在很大程度上解决了以上问题，相比于之前的基因编辑技术，CRISPR-Cas9系统有着一些无可比拟的优点。1.靶点多，基因组中平均8个碱基就能找到一个靶点；2.功能更加丰富，经过不同的改造后可提高编辑效率、及调控基因表达水平；3.CRISPR-Cas9系统构建步骤简单、使用方便。因此CRISPR-Cas9技术能够大大降低实验难度，缩短实验周期，提高效率。
随着生物技术的发展，CRISPR-Cas9系统发展成为能够对多个物种进行精准基因组编辑的技术。CRISPR-Cas9系统是来源于细菌和古生菌中的一种由RNA介导的适应性免疫系统，包括两部分：有切割双链DNA活性的Cas9蛋白和能与靶点DNA序列结合的sgRNA(20个核苷酸)。CRISPR-Cas9系统仅需20bp的RNA介导其对靶点的编辑，该系统存在构建简便快捷、编辑效率高、实验周期短等优点，已经成为目前最受热捧的基因组编辑技术。
此外，因CRISPR-Cas9技术具有简便性和通用性，其在科研、农业、精准医疗领域展现了巨大的应用前景，尤其在传染性疾病、遗传病(如地中海贫血)、肿瘤以及器官移植等领域开辟了全新的途径。
哈佛大学研究人员利用CRISPR-Cas9技术一次性敲除猪细胞中62个逆转录病毒基因，扫清猪器官用于人体移植的重大难关。美国研究人员针利用该技术将艾滋病毒从艾滋病患者的细胞基因组中剔除。中山大学黄军就应用CRISPR-Cas9对人体胚胎细胞中修改β-地中海贫血的基因进行的研究有望治愈这一疾病。
参考文献
[1]:DOI:10.1126/science.aad1191
[2]:DOI:10.1038/srep22555
[3]:DOI:10.1007/s13238-015-0153-5
但是，现有的CRISPR-Cas9系统由两个质粒组成，其中一个质粒表达具有切割双链DNA活性的Cas9蛋白，另一个质粒表达sgRNA scaffold。由于CRISPR-Cas9系统采用的是双质粒系统，存在构建困难，且双质粒易受质粒相容性影响。
酵母菌是酿酒和面包等发酵产品制作过程的主要“参与者”，与人类生产和生活息息相关，广泛应用于食品、医药、化工等领域。酿酒酵母(Saccharomyces.Cerevisiae)在分子遗传学方面是最先作为外源基因表达的酵母宿主物的酵母菌具有比较完备的基因表达调控机制和对表达产物的加工修饰能力。在医药领域酵母也具有得天独厚的优势，如斯坦福大学Christina D.Smolke通过导入来自植物、细菌和啮齿动物的21个基因，成功地在酵母菌内将糖转化为吗啡的前体——蒂巴因(thebaine)，Christina D.Smolke还发现，进一步调整过的酵母可以产生氢可酮——一种由蒂巴因化学合成的止痛药。
发明内容
本发明所要解决的技术问题是克服现有技术的不足，提供一种在一个质粒中表达CRISPR-Cas9系统，减少后续编辑系统构建难度的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体及其应用。
为解决以上技术问题，本发明采用如下技术方案：
本发明的一个目的是提供一种基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体，所述的酿酒酵母基因组编辑载体通过将Cas9蛋白表达框和sgRNA scaffold表达框整合在载体中，获得载体-Cas9-sgRNA scaffold，然后将以ADE1为靶位点设计合成的多条sgRNA片段中的任意一条整合到所述的载体-Cas9-sgRNA scaffold中得到所述的酿酒酵母基因组编辑载体。
具体地，所述的Cas9蛋白表达框包括TEF1启动子、Cas9蛋白、CYC1终止子。
更具体地，所述的Cas9蛋白表达框的序列如SEQ ID NO.1所示。
具体地，所述的sgRNA scaffold表达框包括SNR52启动子、sgRNA scaffold、SUP4终止子。
更具体地，所述的sgRNA scaffold表达框的序列如SEQ ID NO.2所示。
具体地，所述的多条sgRNA片段的序列分别如SEQ ID NO.3、SEQ ID NO.4、SEQ IDNO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8所示。
具体地，所述的载体为pSynoYACO，其序列如SEQ ID NO.9所示。
具体地，所述的载体-Cas9-sgRNA scaffold的序列如SEQ ID NO.10所示。
本发明的另一个目的是提供一种所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体的制备方法，其包括如下步骤：
步骤(1)、根据已知序列设计并合成Cas9蛋白表达框、sgRNA scaffold表达框和多条sgRNA片段；
步骤(2)、将Cas9蛋白表达框重组到经酶切的载体中，获得载体-Cas9；
步骤(3)、将sgRNA scaffold表达框重组到经酶切的载体-Cas9中，获得载体-Cas9-sgRNA scaffold；
步骤(4)、将多条sgRNA片段中的任意一条重组到经酶切的载体-Cas9-sgRNAscaffold中，获得所述的酿酒酵母基因组编辑载体。
本发明中，按照常规基因合成方法合成Cas9蛋白表达框、sgRNA scaffold表达框和多条sgRNA片段，其中，Cas9蛋白表达框分为每段1kb进行合成。
具体地，步骤(2)采用AscI酶切载体；步骤(3)采用PmeI酶切载体-Cas9；步骤(4)采用NotI酶切载体-Cas9-sgRNA scaffold。
具体地，步骤(2)、步骤(3)、步骤(4)中进行重组的反应温度为48～52℃，重组反应时间为50～70min。
具体地，步骤(2)将Cas9蛋白表达框重组到经酶切的载体中，然后电转至Epi300感受态细胞中进行培养，获得所述的载体-Cas9。
具体地，步骤(3)将sgRNA scaffold表达框重组到经酶切的载体-Cas9中，然后电转至Epi300感受态细胞中进行培养，获得所述的载体-Cas9-sgRNA scaffold。
具体地，步骤(4)将多条sgRNA片段中的任意一条重组到经酶切的载体-Cas9-sgRNA scaffold中，然后电转至Epi300感受态细胞中进行培养，获得所述的酿酒酵母基因组编辑载体。
更具体地，步骤(2)、步骤(3)、步骤(4)中电转至Epi300感受态细胞中，然后涂布CmR平板，在36～38℃下进行培养11～12h。
本发明的第三个目的是提供一种基于CRISPR-Cas9系统的酿酒酵母基因组编辑系统的制备方法，其通过将所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体电转至电转化感受态细胞中，然后进行培养得到所述的酿酒酵母基因组编辑系统。
具体地，电转化感受态细胞为VL6-48N电转化感受态细胞。
VL6-48N电转化感受态细胞制备方法：
1.取超低温冰箱中冻存的VL6-48N酵母菌液于YPD平板上稀释涂布，30℃培养2天，获得单菌落；
2.挑取单菌落于50mLYPD液体培养基中培养过夜；
3.当菌液生长到OD600在0.4-0.6时3000rpm离心10min收集菌体；
4.0℃预冷的无菌水重悬菌体，3000rpm离心10min；
5.0℃预冷的10％甘油重悬菌体，3000rpm离心10min；
6.重复步骤5；
7.1mL10％甘油重悬步骤6菌体，每管200uL菌液，分装于1.5mL无菌EP管中，即为VL6-48N电转感受态。
本发明的第四个目的是提供一种所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体在酿酒酵母基因中的应用。
由于上述技术方案的实施，本发明与现有技术相比具有如下优点：
本发明采用单质粒并单拷贝的载体系统，采用一个质粒同时表达Cas9蛋白和sgRNA scaffold(能在两种不同的生物中复制的载体，例如既能在原核生物中复制,又能在真核生物中复制的载体)，使得靶基因编辑系统只需一步构件和转化，操作简便。且本发明构建的质粒为单拷贝，属于严谨性控制。
说明书附图
图1为pSynoYACO载体图；
图2为pSynoYACO-Cas9-sgRNA scaffold载体图。
具体实施方式
下面结合具体实施例对本发明做进一步详细的说明，但本发明并不限于以下实施例。本发明中若无特别说明，原料均可市购获得，方法为本领域的常规方法。
实施例1：
1、根据已知序列设计以下序列：
1)酵母Cas9蛋白表达框(TEF1promoter-Cas9-CYC1terminitor)，包括TEF1启动子，Cas9蛋白，CYC1终止子，序列见SEQ ID NO.1，其中：位于SEQ ID NO.1序列首尾的CGAACGCCATCGACTTACCAGTATGCTACTTACTAT和CAGCAGGAGCTGGACTCTACTGATGTCTGGACAGC为Cas9蛋白表达框与pSynoYACO载体同源臂序列；ggcgcgcc和ggcgcgcc为AscI酶切位点序列，GTTTAAAC为PmeI酶切位点。
2)sgRNA scaffold表达框(SNR52 promoter+sgRNA Scaffold+SUP4terminitor)序列见SEQ ID NO.2，其中：位于SEQ ID NO.2序列首尾的GGACGCTCGAAGGCTTTAATTTGC和gctgctaacaaagcccgaaag为sgRNA Scaffold表达框与pSynoYACO-Cas9载体同源臂序列，GTTTAAAC为PmeI酶切位点序列。
3)以ADE1为靶位点的6条sgRNA片段的引物，序列见SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8。
ADE1sgRNA片段与pSynoYACO-Cas9-sgRNA scaffold同源臂序列为
F：tctccgcagtgaaagataaatgatc(SEQ ID NO.11)
R：GTTTTAGAGCTAGAAATAGCAAGTT(SEQ ID NO.12)。
2、根据常规基因合成方法合成步骤1中所有片段。
1)PCR扩增
其中Cas9蛋白表达框分为每段1kb进行合成，一轮和二轮反应体系如下表。
表1为PCR扩增一轮反应体系，表2为PCR扩增一轮反应程序。
表1
表2
一轮反应结束后进行二轮反应。
表3为PCR扩增二轮反应体系，表4为PCR扩增二轮反应程序。
表3
| 组分 | 用量(μl) |
| ddH2O | 35 |
| 5×PCR | 10 |
| 10mM dNTP | 1 |
| 一轮反应产 | 1 |
| Primer F | 1 |
| Primer R | 1 |
| S15酶 | 1 |
表4
经二轮扩增得到的PCR产物进行胶回收。
2)平连反应：
胶回收片段进行平连反应，反应体系如下表5，其中平连载体为经平末端内切酶酶切后的pUC载体。
表5
| 组分 | 用量(μl) |
| 2×QB | 5 |
| PCR产物 | 3 |
| 平连载体 | 1 |
| T4连接酶 | 1 |
平连反应程序为22℃反应30min。
3)转化、涂板
将上述反应液冷却后加入到于冰上融化的感受态细胞中，轻弹管壁数下混匀，冰浴放置30min。42℃热激90秒，冰浴2min。加入400～500μL LB培养基，37℃恢复培养60min。5000rpm离心5min后弃去上清，沉淀重悬后均匀涂布筛选平板上。于37℃培养12h。
4)克隆鉴定
用无菌牙签挑选单菌落于200μl左右LB培养基中，放入37℃摇床1～2h后，取3μl菌液进行菌检PCR扩增【菌检引物为77：GATGTGCTGCAAGGCGATTA(SEQ ID NO.13)和88：TTATGCTTCCGGCTCGTATG(SEQ ID NO.14)】，剩余菌液继续放入摇床。6h后将检到的菌液送测2～4个。表6为菌检反应体系，表7为菌检反应程序。
表6
表7
菌检反应结束后，点胶验证PCR产物大小是否正确，培养6h后将检到的菌液送测2～4个，测序引物同样为77/88。
将测序结果与设计序列进行比对，确定正确克隆。
以正确克隆质粒为模板进行扩增获得序列正确PCR产物，各PCR片段进行PCR反应拼接，获得拼接产物，拼接产物胶回收。
Cas9蛋白表达框(TEF1promoter-Cas9-CYC1terminitor)片段，其与pSynoYACO载体AscI酶切位点两侧同源臂为CGAACGCCATCGACTTACCAGTATGCTACTTACTAT(SEQ ID NO.15)和GCTGTCCAGACATCAGTAGAGTCCAGCTCCTGCTG(SEQ ID NO.16)(其与SEQ ID NO.1的同源臂反向互补)，同时也是Cas9蛋白表达框扩增引物。sgRNA scaffold表达框与pSynoYACO-Cas9载体同源臂为GGACGCTCGAAGGCTTTAATTTGC(SEQ ID NO.17)和CTTTCGGGCTTTGTTAGCAGC(SEQID NO.18)(其与SEQ ID NO.2的同源臂反向互补)，同时也是其扩增引物。sgRNA序列两端为sgRNA片段与pSynoYACO-Cas9-sgRNA scaffold载体NotI酶切位点两侧的同源臂为tctccgcagtgaaagataaatgatc(SEQ ID NO.11)和GTTTTAGAGCTAGAAATAGCAAGTT(SEQ IDNO.12)。
3、AscI酶切载体pSynoYACO(SEQ ID NO.9)，采用切胶回收酶切后的载体。
4、TEF1promoter-Cas9-CYC1terminitor片段重组经AscI酶切的pSynoYACO载体片段，50℃反应1h。反应结束后转化Epi300感受态细胞，涂布CmR平板。37℃培养12h后挑取单克隆进行菌检，挑取菌检大小正确克隆进行送测，测序结果与序列进行比对，获得pSynoYACO-Cas9正确克隆。
5、PmeI酶切pSynoYACO-Cas9载体正确克隆，采用切胶回收酶切后的载体。
6、sgRNA scaffold表达框片段重组经PmeI酶切的pSynoYACO载体片段，50℃反应1h。反应结束后转化Epi300感受态细胞，涂布CmR平板。37℃培养12h后挑取单克隆进行菌检，挑取菌检大小正确克隆进行送测，测序结果与序列进行比对，获得pSynoYACO-Cas9-sgRNA scaffold正确克隆(SEQ ID NO.10)。
7、NotI酶切pSynoYACO-Cas9-sgRNA scaffold载体正确克隆，采用切胶回收酶切后的载体。
8、sgRNA片段重组经NotI酶切的pSynoYACO-Cas9-sgRNA scaffold载体片段，50℃反应1h。反应结束后转化Epi300感受态细胞，涂布CmR平板。37℃培养12h后挑取单克隆进行菌检，挑取菌检大小正确克隆进行送测，测序结果与序列进行比对，获得pSynoYACO-Cas9-ADE1-sgRNA scaffold正确克隆。
9、制备VL6-48N电转化感受态细胞：
(1)取超低温冰箱中冻存的VL6-48N酵母菌液于YPD平板上稀释涂布，30℃培养2天，获得单菌落；
(2)挑取单菌落于50mLYPD液体培养基中培养过夜；
(3)当菌液生长到OD600在0.4-0.6时3000rpm离心10min收集菌体；
(4)0℃预冷的无菌水重悬菌体，3000rpm离心10min；
(5)0℃预冷的10％甘油重悬菌体，3000rpm离心10min；
(6)重复步骤5；
(7)1mL10％甘油重悬步骤6菌体，每管200uL菌液，分装于1.5mL无菌EP管中，即为VL6-48N电转感受态。
10、电转1ug pSynoYACO-Cas9-ADE1-sgRNA scaffold载体。
11、电转后菌液于含有YPD液体培养基的单管中培养12h。
12、3000rpm，2min离心上述菌液，山梨醇清洗两次细胞，涂布0.06g/L腺嘌呤的组氨酸缺陷型酵母固体培养基。
13、2天后观察酵母单克隆生长情况，统计红色克隆比例。
14、挑取红色克隆于单管中进行培养30℃，1天。
15、将上述菌液提取基因组。
采用ADE1扩增引物CAATTACGAAGACTGAACTGGACGG(SEQ ID NO.19)和CTACGTGACAAATCTTCACCCACCAG(SEQ ID NO.20)扩增提取出的酵母基因组并对PCR产物进行测序。分析测序结果，计算成功编辑的克隆比例30％。
以上对本发明做了详尽的描述，其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，且本发明不限于上述的实施例，凡根据本发明的精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。
Claims (10)
1. 一种基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体，其特征在于：所述的酿酒酵母基因组编辑载体通过将Cas9蛋白表达框和sgRNA scaffold表达框整合在载体中，获得载体- Cas9-sgRNA scaffold，然后将以ADE1为靶位点设计合成的多条sgRNA片段中的任意一条整合到所述的载体- Cas9-sgRNA scaffold中得到所述的酿酒酵母基因组编辑载体。
2.根据权利要求1所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体，其特征在于：所述的Cas9蛋白表达框包括TEF1启动子、Cas9蛋白、CYC1终止子。
3. 根据权利要求2所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体，其特征在于：所述的Cas9蛋白表达框的序列如SEQ ID NO.1所示。
4. 根据权利要求1所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体，其特征在于：所述的sgRNA scaffold表达框包括SNR52启动子、sgRNA scaffold、SUP4终止子。
5.根据权利要求4所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体，其特征在于：所述的sgRNA scaffold表达框的序列如SEQ ID NO.2所示。
6. 根据权利要求1所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体，其特征在于：所述的多条sgRNA片段的序列分别如SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQID NO.6、SEQ ID NO.7、SEQ ID NO.8所示。
7. 根据权利要求1所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体，其特征在于：所述的载体为pSynoYACO，其序列如SEQ ID NO.9所示。
8.一种如权利要求1至7中任一项所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体的制备方法，其特征在于：其包括如下步骤：
步骤（1）、根据已知序列设计并合成Cas9蛋白表达框、sgRNA scaffold表达框和多条sgRNA片段；
步骤（2）、将Cas9蛋白表达框重组到经酶切的载体中，获得载体-Cas9；
步骤（3）、将sgRNA scaffold表达框重组到经酶切的载体-Cas9中，获得载体- Cas9-sgRNA scaffold；
步骤（4）、将多条sgRNA片段中的任意一条重组到经酶切的载体- Cas9-sgRNAscaffold中，获得所述的酿酒酵母基因组编辑载体。
9.一种基于CRISPR-Cas9系统的酿酒酵母基因组编辑系统的制备方法，其特征在于：其通过将权利要求1至7中任一项所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体电转至电转化感受态细胞中，然后进行培养得到所述的酿酒酵母基因组编辑系统。
10.一种如权利要求1至7中任一项所述的基于CRISPR-Cas9系统的酿酒酵母基因组编辑载体在酿酒酵母基因中的应用。
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