CN107858346A - 一种敲除酿酒酵母染色体的方法 - Google Patents
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Abstract
本发明涉及生物技术领域,具体公开了一种敲除酿酒酵母染色体的方法。本发明利用CRISPR/Cas9基础技术切除酵母完整染色体,通过在着丝粒附近选择特异性靶点,设计对应的guide RNA引导Cas9蛋白在染色体着丝粒附近产生切口,从而实现整条染色体的敲除。本发明与现有通过Gal启动子诱导、减数分裂等方式实现整条染色体丢失的方法相比,更加简单、高效、快速地实现酿酒酵母染色体的切割:避免姐妹染色单体的交叉互换,获得染色体敲除后的酿酒酵母纯合二倍体菌株。
Description
技术领域
本发明涉及生物技术领域,更具体的说是涉及一种敲除酿酒酵母染色体的方法。
背景技术
生物基因组携带了决定生物基本性状的遗传信息,人工DNA合成技术和DNA大片段操作技术推动了基因组人工合成研究的进步。合成生物学的发展推动了通过人工设计合成来“写”基因组信息标志着“人造生命”的开始。
基因组DNA长度过大,而且绝大多数真核生物具有多条染色体,且染色体长度都较大,涉及到染色体疾病或以染色体为单位的功能整合等都需要对整条染色体进行操作。如何实现整条染色体的敲除是一个值得探讨的问题。酿酒酵母是与人类关系最广泛的一种酵母,它用于制作面包和馒头等食品以及酿酒,同时还可以作为微生物发酵生产化学品的工程菌。酿酒酵母有单倍体和双倍体两种生活形态,由于双倍体酿酒酵母营养细胞大,生活能力强,工业上多用双倍体进行生产。为了能够进一步提高酿酒酵母的生产能力,需要对酵母的基因组进行改造,如果直接用二倍体改造,可能得到优良菌株是杂合的,遗传性状不稳定,而单倍体就成为一种很好地选择。通过将单倍体酵母改造为一种优良菌株,而后再通过交配重新形成优势的二倍体。
但是,酿酒酵母在形成二倍体的过程中,有一定几率发生姐妹染色单体的交叉互换,还是会形成杂合二倍体。因此,对不需要的染色体及时进行敲除,能够避免这种问题的出现(单倍体酵母染色体因含有大量必需基因,无法直接敲除,必须在二倍体状态下敲除才能够存活)。
在酿酒酵母中,还未发现对整条染色体的敲除技术。目前在酵母中常用的敲除技术是基因敲除技术,它是以达到定点修饰改造染色体上某一基因的目的的一种技术,仅能对基因进行操作。现有利用减数分裂时酵母染色体染色体的不均等分配来实现染色体的丢失的方法,在减数分裂过程中,酵母的同源染色体之间会发生同源重组,不能保证单条染色体的完整性。通过这种方法要得到完整染色体的工作量大且效率低。
发明内容
有鉴于此,本发明的目的在于提供一种敲除酿酒酵母染色体的方法,使得所述方法能够提高敲除整条染色体的效率,达到80%以上的敲除率,避免姐妹染色单体交叉互换对染色体的影响,确保酿酒酵母整条染色体的敲除,获得染色体敲除后的纯合二倍体酵母。
为实现上述发明目的,本发明提供如下技术方案:
步骤1、在待敲除的染色体着丝粒左右100bp范围内寻找PAM序列作为guide RNA靶位点,以PAM序列上游20个碱基序列作为protospacers序列,所述PAM序列和protospacers序列不存在于与待敲除染色体的单倍体酿酒酵母交配的酿酒酵母对应的同源染色体上;
步骤2、在protospacers序列两端构建载体同源臂序列,并形成完全互补的双链DNA,通过酶切和Gibson组装技术,与所述载体组装,获得guide RNA质粒;
步骤3、将待敲除染色体的单倍体酿酒酵母菌株与交配型不同得酿酒酵母菌株进行融合,构建二倍体酿酒酵母菌株,向二倍体酿酒酵母细胞中转化Cas9质粒和guide RNA质粒,对待敲除的染色体着丝粒切割,敲除整条染色体;或
向与待敲除染色体的单倍体酿酒酵母交配的酿酒酵母中转化Cas9质粒和guideRNA质粒,然后与待敲除染色体的单倍体酿酒酵母菌株进行融合,构建二倍体酿酒酵母菌株,并对待敲除的染色体着丝粒切割,敲除整条染色体。整体流程示意图见图1。
针对现有利用Gal启动子诱导、减数分裂使酵母染色体染色体的不均等分配来实现染色体丢失的方法工作量大、效率低的缺陷,本发明依靠CRISPR/Cas9基础技术,选择着丝粒附近的guide RNA识别位点,由guide RNA引导Cas9蛋白在染色体着丝粒附近产生切口,从而实现整条染色体的敲除,整个方法更稳定有效,成功率更高。
作为优选,所述载体同源臂为载体上酶切位点两端的同源臂;其中,更优选地,所述载体为pRS42H质粒,而所述酶切位点选择NotI酶切位点,质粒图谱见图3。在本发明具体实施方式中,本发明选择pRS42H质粒作为载体,同时选择其上NotI酶切位点两端的同源臂序列,故本发明步骤2可具体为:
在protospacers序列两端,构建pRS42H质粒上NotI酶切位点两端同源臂序列,然后形成完全互补的双链DNA,通过酶切和Gibson组装技术,与pRS42H质粒组装,获得guideRNA质粒。
更为具体地,所述pRS42H质粒上NotI酶切位点两端同源臂序列如SEQ ID NO:3和4所示。其中,SEQ ID NO:3所示序列为上游同源臂序列:GCAGTGAAAGATAAATGATC;SEQ ID NO:4所示序列为下游同源臂序列:GTTTTAGAGCTAGAAATAGC。在更加具体的实施过程中,在protospacers序列两端构建载体同源臂序列,并形成完全互补的双链DNA操作,以上述SEQID NO:3和4所示序列为例,可以是GCAGTGAAAGATAAATGATC+protospacers序列+GTTTTAGAGCTAGAAATAGC和GCTATTTCTAGCTCTAAAAC+protospacers序列+GATCATTTATCTTTCACTGC,两者完全互补,通过退火粘合,形成双链DNA。
本发明所述Cas9质粒可按照常规方法合成,如利用引物PCR扩增表达Cas9的基因,引物两端带酶切位点,利用酶切连接将片段与酶切后的载体构建为完整质粒,在本发明的具体实施方式中,所述载体选择为pRS415质粒,构建后的Cas9质粒图谱见图4。
根据本发明的技术方案,本发明以敲除synIII人工酿酒酵母合成型III号染色体为例进行了举例说明,则本发明步骤1为:
synIII人工酿酒酵母在BY4742(商业化酵母)基础上,III号染色体全人工合成。在synIII人工酿酒酵母合成型III号染色体的着丝粒左右100bp范围内寻找PAM序列作为guide RNA靶位点,以PAM序列上游20个碱基序列作为protospacers序列,所述PAM序列和protospacers序列不存在于与synIII人工酿酒酵母交配的BY4741酿酒酵母(商业化酵母)对应的同源染色体上。
其中,所述PAM序列如SEQ ID NO:1所示,所述protospacers序列如SEQ ID NO:2所示,SEQ ID NO:2+SEQ ID NO:1即为确定的guide RNA可识别点序列:ttatacgaagttattataagCGG(大写碱基即为PAM序列,小写碱基为protospacers序列)。
本发明以敲除synIII人工酿酒酵母合成型III号染色体为例,经过Tester a和Tester alpha初步验证敲除效率,敲除效率达83%,达到了极高水平。
由以上技术方案可知,本发明利用CRISPR/Cas9技术,从待敲除的整条染色体的着丝粒附近选择guide RNA可识别位点,从而使guide RNA质粒引导Cas9蛋白在着丝粒附近发挥作用,使酿酒酵母染色体着丝粒附近产生切口,实现酿酒酵母整条染色体的简单、高效、快速和完整敲除,同时可避免姐妹染色单体的交叉互换,获得染色体敲除后的酿酒酵母为纯合二倍体菌株,也能够为酿酒酵母整合16条合成染色体提供技术指导。
附图说明
图1所示为以敲除synIII人工酿酒酵母合成型III号染色体为例的流程示意图;
图2所示为敲除合成型III号染色体后PCR验证凝胶电泳图;
图3所示为pRS42H质粒图谱;
图4所示为Cas9质粒图谱。
具体实施方式
本发明公开了一种敲除酿酒酵母染色体的方法,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明所述方法已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述启动子和菌株进行改动或适当变更与组合,来实现和应用本发明技术。
在本发明技术方案中,可以在适当的载体、基因元件中插入筛选标签(如氨基酸营养标签或抗性标签),用于敲除过程中对正确菌株的筛选验证,也可采用本领域其他方式进行验证。
下面结合实施例,进一步阐述本发明。
实施例1:敲除synIII人工酿酒酵母合成型III号染色体
1、将待敲除合成III号染色体(synIII)的synIII人工酿酒酵母yYW0233(MATα,BY4742基础上III号染色体全人工合成)菌株与含野生III号染色体(wtIII)的yYW0171(MATa,即BY4741酿酒酵母)菌株对比,在synI II着丝粒左右100bp范围内附近找到guide RNA可识别位点gRNA-synIII ce n-L“ttatacgaagttattataagCGG”,其中CGG为PAM序列,ttatacgaagttattataag为protospacers序列。
2、构建guide-RNA质粒,其构建步骤如下:
选择protospacers为ttatacgaagttattataag,采用pRS42H质粒的NotI酶切位点作为插入点;
人工合成引物“GCAGTGAAAGATAAATGATCttatacgaagttattataagGTTTTAGAGCTAGAAATAGC”和“GCTATTTCTAGCTCTAAAACcttataataacttcgtataaGATCATTTATCTTTCACTGC”;其中,大写碱基为pRS42H质粒的NotI酶切位点上、下游同源臂,小写碱基即为protospacers序列,两个引物完全互补;
退火粘合两个引物,得到双链DNA;
利用限制性内切酶NotI和CIP(防止载体质粒自连)消化质粒pRS42H(含Hygromycin抗性标签),使之线性化;利用Gibson组装将线性化质粒和双链DNA进行组装;
将反应体系转化如DH5α大肠杆菌感受态细胞中,涂布于LB+Carb平板上,37℃培养12h;
挑取5个单菌落接种于5mL LB+Carb液体培养基中,37℃过夜培养后,提取质粒,进行Sanger测序;
测序正确的菌株命名为bYW0138,作为承载guide-RNA质粒的菌株。
3、yYW0171(MAT a)与synIII人工酿酒酵母yYW0233(MAT α)进行杂交,把两种酵母菌平板划线活化后,同时接种到5ml YPD液体培养基中,30℃过夜培养后,将菌液划线到YPD平板上,利用酵母显微操作仪,手动挑取酿酒酵母二倍体细胞,或者通利用两种酵母细胞杂交后营养缺陷型的互补,30℃培养2-3d后,涂布SC缺陷型培养基筛选出阳性杂交细胞。待显微镜挑出或平板筛选出的细胞在30℃长出单菌落,用菌落PCR法验证该二倍体细胞的交配型,若同时具有MAT a和MAT α两种交配型,并且合成型III号染色体和野生型III号染色体的PCRtag都在,则证明杂交成功,挑选出表型稳定的一株,将正确菌株命名为yYW0234。
向二倍体菌株yYW0234中转化Cas9质粒(含有Leu筛选标签),在SC-Leu培养平板上筛选。得到的菌株转化guide RNA质粒,在SC-Leu+Hygr omycin B培养平板上筛选。然后挑取SC-Leu+Hygromycin B培养平板上生长的单菌落在SC-Leu+Hygromycin B培养平板上划线分纯,此时得到的菌株为丢失synIII染色体的酿酒酵母二倍体菌株。
在SD培养平板表面分别涂布Tester a和Tester alpha两种菌(Tester a和Testeralpha是两种验证菌株,在涂布这两种菌株的平板上,只有交配型相反且单一的菌株才能够生长,因为酿酒酵母的三号染色体是性染色体,敲除后为单一性别,如果没有敲除的话,两个平板都不长),将SC-Leu+Hygromy cin B培养平板上的菌翻印到两个板,能初步验证合成型III号染色体的丢失,在Tester alpha上生长菌的数量除以SC-Leu+Hygromycin B培养平板上菌的数量,计算得出敲除效率为83%。
也可利用PCR反应进行每株菌验证,例如挑取一株二倍体酿酒酵母菌株提取基因组并使用GoTaq Green Master Mix利用PCR反应进行验证。利用特异性区分synIII和wtIII的引物进行PCR反应,结果显示存在染色体wtIII,而不存在synIII染色体,证明成功敲除了整条染色体,将此菌株命名为yYW0235,结果见图2。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 天津大学
<120> 一种敲除酿酒酵母染色体的方法
<130> MP1701573
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cgg 3
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ttatacgaag ttattataag 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcagtgaaag ataaatgatc 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gttttagagc tagaaatagc 20
Claims (10)
1.一种敲除酿酒酵母染色体的方法,其特征在于,包括:
步骤1、在待敲除的染色体着丝粒左右100bp范围内寻找PAM序列作为guide RNA靶位点,以PAM序列上游20个碱基序列作为protospacers序列,所述PAM序列和protospacers序列不存在于与待敲除染色体的单倍体酿酒酵母交配的酿酒酵母对应的同源染色体上;
步骤2、在protospacers序列两端构建载体同源臂序列,并形成完全互补的双链DNA,通过酶切和Gibson组装技术,与所述载体组装,获得guide RNA质粒;
步骤3、将待敲除染色体的单倍体酿酒酵母菌株与交配型不同得酿酒酵母菌株进行融合,构建二倍体酿酒酵母菌株,向二倍体酿酒酵母细胞中转化Cas9质粒和guide RNA质粒,对待敲除的染色体着丝粒切割,敲除整条染色体;或
向与待敲除染色体的单倍体酿酒酵母交配的酿酒酵母中转化Cas9质粒和guide RNA质粒,然后与待敲除染色体的单倍体酿酒酵母菌株进行融合,构建二倍体酿酒酵母菌株,并对待敲除的染色体着丝粒切割,敲除整条染色体。
2.根据权利要求1所述方法,其特征在于,所述载体同源臂为载体上酶切位点两端的同源臂。
3.根据权利要求2所述方法,其特征在于,所述酶切位点为NotI酶切位点。
4.根据权利要求2所述方法,其特征在于,所述载体为pRS42H质粒。
5.根据权利要求1所述方法,其特征在于,所述Cas9质粒由以下方法构建获得:
利用引物PCR扩增表达Cas9的基因,引物两端带酶切位点,利用酶切连接将片段与酶切后的载体构建为完整质粒。
6.根据权利要求1所述方法,其特征在于,步骤1为:
在synIII人工酿酒酵母合成型III号染色体的着丝粒左右100bp范围内寻找PAM序列作为guide RNA靶位点,以PAM序列上游20个碱基序列作为protospacers序列,所述PAM序列和protospacers序列不存在于与synIII人工酿酒酵母交配的Yyw0171酿酒酵母对应的同源染色体上。
7.根据权利要求6所述方法,其特征在于,所述PAM序列如SEQ ID NO:1所示。
8.根据权利要求6所述方法,其特征在于,所述protospacers序列如SEQ ID NO:2所示。
9.根据权利要求1所述方法,其特征在于,步骤2为:
在protospacers序列两端,构建pRS42H质粒上NotI酶切位点两端同源臂序列,然后形成完全互补的双链DNA,通过酶切和Gibson组装技术,与pRS42H质粒组装,获得guide RNA质粒。
10.根据权利要求9所述方法,其特征在于,所述pRS42H质粒上NotI酶切位点两端同源臂序列如SEQ ID NO:3和4所示。
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