CN108588128A - 一种高效率大豆CRISPR/Cas9***的构建方法及应用 - Google Patents
一种高效率大豆CRISPR/Cas9***的构建方法及应用 Download PDFInfo
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Abstract
本发明公开了一种高效率大豆CRISPR/Cas9***的构建方法及应用,将Staphylococcus aureus Cas9蛋白进行拆分表达,为了提高对大豆基因组的编辑效率及广谱性,构建了大豆SaCas9编辑***。本发明将一个低效率的编辑***转变为高效率编辑***,不仅拓展了编辑大豆基因组的应用广度,也为今后解决农作物基因组编辑效率的问题上提供新思路。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及一种高效率大豆CRISPR/Cas9***的构建方法及应用。
背景技术
CRISPR/Cas9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,可用来对抗入侵的病毒及外源DNA。CRISPR/Cas9***通过将入侵噬菌体和质粒DNA的片段整合到CRISPR中,并利用相应的CRISPR RNAs(crRNAs)来指导同源序列的降解,从而提供免疫性。
这个发现纯属偶然,起初也没有引起包括发现者本身太大的重视,甚至这种特征序列都没有一个名字,直到2002年,在通过计算机操作发现很多原核生物(真细菌和古细菌),都有类似被21-37bp的回文重复序列间隔开的非重复序列后,才正式有了这个顾名思义的名字CRISPR,成簇的、规律间隔的、短回文、重复序列(clustered regularlyinterspaced short palindromic repeats,CRISPR),并且除了这样的特征序列外,其附近还有一段CRISPR-associated基因,也就是我们后来说的发挥剪切作用和整合外源片段作用的一系列Cas蛋白,CRISPR***中最重要的3大元件—spacer,repeats,cas等。目前,已经在48%的真细菌和95%的古细菌中发现了CRISPR***,可以算得上是普遍存在了。
CRISPR/Cas9***不但丰富了我们对于细菌、古细菌生理机制的认知,更重要的是这种体系的改造利用能带来席卷整个分子生物学领域,更新现有操作模式的一场技术革命。同时,它也为我们打开了一扇窗,从CRISPR的角度重新认识整个微生物世界自我和相互间的调控网络、调控机制,原核及真核细胞间的异同和联系,甚至协同进化的证据。
现在使用的CRISPR/cas 9***是由最简单的type II CRISPR改造而来的DNA定点剪切工具,该***由单链的guide RNA和有核酸内切酶活性的Cas 9蛋白构成。通过cas9蛋白形成DNA双链的断裂,而细胞通过NHEJ的修复会造成INDEL效应(insertion anddeletion),进而造成基因的移码突变而达到基因敲除的目的。此外,还可以通过同源重组等方式达到对基因精确编辑的目的。其实,在此之前已经发展了多种基因的编辑工具,如:ZFN,TALEN等,并且也已经得到广泛应用。CRISPR可谓是2013年生物界的焦点,这项技术相对与ZFN,TALEN等基因编辑技术可以说是既简便又经济,一般的实验室都可以构建自己的平台。
大豆(Glycine max(Linn.)Merr.)是世界重要的粮油兼用作物,也是人类优质蛋白的主要来源。既是我国主要农作物之一,也是我国进口量最大的农产品。大豆是关系国计民生的重要性物资,又是最具经济效益的作物,其延长的产业链和价值链具有很大发展潜力,在农产品贸易领域扮演举足轻重的角色。大豆是人类不可或缺的高蛋白食品、健康植物油及重要保健品的原料。其籽粒含有丰富的蛋白质及脂肪,两者约占干重的60%。大豆是蛋白质含量最高的主要作物,蛋白质一般含量40%,高者达50%。大豆含油量一般18%,高者24%,为世界提供了30%的脂肪和60%的植物蛋白质。
目前运用于大豆基因组编辑的工具甚少,远远满足不了我们对多类型位点且高效率编辑的需求。据已报导的数据显示,2015年SpCas9已经被运用于大豆子叶瞬时转化和稳定遗传转化体系中,但是编辑效率还有待进一步优化。而2016年Cpf1也被运用于大豆原生质体中,但是未在稳定遗传转化体系中验证其编辑效率。因此,开发更高效率更多选择更广运用的CRISPR/Cas9体系,并将其运用于大豆基因组编辑迫在眉睫。
发明内容
鉴于上述现有技术中存在的缺陷,本发明的目的是提出一种高效率大豆CRISPR/Cas9***的构建方法及应用。
为了实现上述目的,本发明采用了如下技术方案:
一种高效率大豆CRISPR/Cas9***的构建方法,包括以下步骤:
步骤1)人工合成大豆特异的U6启动子序列、Staphylococcus aureus Cas9序列和Streptococcus pyogenes Cas9序列;
步骤2)将扩增目的片段GmU6,sgRNA,35S,N-SpCas9,C-SpCas9,Bar片段构建于实验室改造的pCAMBIA1300骨架载体上,获得以下载体骨架:
GmU6::sgRNA-35S::SpCas9-35S::Bar
GmU6::sgRNA-35S::SaCas9-35S::Bar
GmU6::sgRNA-35S::N-SpCas9-35S::C-SpCas9-35S::Bar
GmU6::sgRNA-35S::N-SaCas9-35S::C-SaCas9-35S::Bar;
步骤3)从SoyBase中选择3个大豆基因以及2个大豆gma-miRNA,根据选定的基因以及miRNA设计靶位点序列,合成两段带有BsaI酶切后形成的粘性末端互补序列的引物,长度为19bp-21bp;
步骤4)将sgRNA靶位点引物退火后形成的带有粘性末端的核心序列,组装于步骤2)所构建的载体;
步骤5)获得完整的大豆CRISPR/Cas9编辑载体。
一种根据上述的高效率大豆CRISPR/Cas9***在大豆基因组修饰中的应用。包括以下步骤:
(1)将上述获得的大豆CRISPR/Cas9编辑载体转化K599农杆菌;
(2)侵染大豆子叶,培养获得瞬时转化的大豆根毛;
(3)运用CTAB法提取大豆根毛DNA,测序检测编辑效率。
本发明的突出效果为:
本发明的一种高效率大豆CRISPR/Cas9***的构建方法及应用,将Staphylococcus aureus Cas9蛋白进行拆分表达,为了提高对大豆基因组的编辑效率及广谱性,构建了大豆SaCas9编辑***。由于植物体内可能存在着对CRISPR/Cas9编辑***的免疫作用,完整的SaCas9蛋白编辑效率远远低于蛋白编辑效率,而且完整的SaCas9蛋白***对于大豆可能有毒害作用,相对于***,完整SaCas9***的大豆瞬转根毛生长周期长,根毛生长均较短,状态不佳。本发明将一个低效率的编辑***转变为高效率编辑***,不仅拓展了编辑大豆基因组的应用广度,也为今后解决农作物基因组编辑效率的问题上提供新思路。
附图说明
图1为本发明实施例1的SaCas9瞬转大豆根毛生长情况;
图2A为本发明实施例1中构建的载体结构;
图2B为本发明实施例1中splitSaCas9编辑gma-miR156b的突变类型;
图2C为本发明实施例1中splitSaCas9编辑Glyma02g47021的突变类型及编辑效率;
图2D为本发明实施例1中splitSaCas9编辑Glyma14g基因的突变类型及编辑效率。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
实施例1:
本实施例所用试剂和材料的来源:
1)pCAMBIA1300载体为实验所有;
2)引物合成及测序均委托北京擎科新业生物技术有限公司;
3)大豆品种为YC03-3;
4)构建载体所用试剂为Vazyme ClonExpress无缝克隆试剂盒,NEB T4ligase,TIANAprep Mini Plasmid Kit II质粒提取试剂盒,TaKaRa MiniBest DNA FragmentPurification Kit,NEB BsaI酶等。
一.大豆CRISPR/Cas9编辑载体的构建
1、挑取pCAMBIA1300质粒单菌落,接种于50ml含卡那抗生素的液体LB培养基中,置于摇床37℃,220rpm/r培养过夜;
2、离心收集菌体,用TIANAprep Mini Plasmid Kit II质粒提取试剂盒提取质粒;
3、选择合适的酶切位点,酶切pCAMBIA1300质粒,于37℃1h后用TaKaRa MiniBestDNA Fragment Purification Kit试剂盒回收线性质粒片段,备用;
4、扩增目的片段GmU6,sgRNA,35S,N-SpCas9,C-SpCas9,Bar片段,随后用VazymeClonExpress无缝克隆试剂盒进行重组连接,构建以下4类载体,如图2A所示:
GmU6::sgRNA-35S::SpCas9-35S::Bar
GmU6::sgRNA-35S::SaCas9-35S::Bar
GmU6::sgRNA-35S::N-SpCas9-35S::C-SpCas9-35S::Bar
GmU6::sgRNA-35S::N-SaCas9-35S::C-SaCas9-35S::Bar
获得载体后分别转化大肠杆菌Top10,37℃培养箱培养过夜,第二天挑取阳性单克隆测序;
5、用NEB BsaI酶切所构建好的质粒,于37℃酶切1h,用TaKaRa MiniBest DNAFragment Purification Kit纯化回收片段;
6、从SoyBase中选取3个与固氮相关的大豆基因Glyma14G146700、Glyma12G221500、Glyma01g198100以及大豆gma-miR156b、gma-miR156c为编辑对象,设计靶点引物(Spcas9选择NGG上游20bp的序列为靶点,Sacas9选择NNGAGT上游21bp的序列为靶点),将sgRNA靶位引物退火后形成的带有粘性末端的核心序序列,用T4链接酶,16℃连接过夜,组装于步骤5所构建的载体(根据载体连接相应的靶点序列),所用引物如表1:
表1
7、将获得的目的载体转化大肠杆菌Top10菌株,于37℃培养箱培养过夜,第二天挑取阳性单克隆测序,质粒保存于-20℃备用,从而获得完整的大豆CRISPR/Cas9编辑载体。
二.大豆基因组编辑效率的检测
1、将表2中所构建的大豆编辑载体分别转化K599农杆菌,质粒按1:50加入K599农杆菌中,置于液氮中1min,37℃冰浴5min,42℃热激5min,最后置于冰上3min后加入新鲜的无抗性LB液体培养基,28℃,220rpm/r培养3h;
表2
2、用大豆子叶发根法检测该Sacas9体系的活性:
试验采用Cho等人发表的高效率大豆根毛转化程序(Cho,H.J.,S.K.Farrand,G.R.Noel and J.M.Widholm(2000)."High-efficiency induction of soybean hairyroots and propagation of the soybean cyst nematode."Planta 210(2):195-204.),并按照应珊和王秀荣等人(应珊,何晓薇,王秀荣and寿惠霞(2008)."影响农杆菌介导的大豆转化效率的因素研究."分子植物育種6(1):32-40.)根据国内条件和实验经验进行进一步优化。
(1)种子消毒:种子采用干燥表面消毒
将成熟的大豆种子单层排列在培养皿中,将装有培养皿的干燥器放到通风橱中,并将所有的培养皿打开,中间放置一个250mL的烧杯。在烧杯中加入100mL的次氯酸钠,然后沿着杯壁缓缓加入4.2mL12mol/L HCl,立即盖上干燥器的盖子,静置过夜(13-14hr)后,盖上培养皿并将其放到超净工作台中,再将培养皿打开大约30min以便除去过多的氯气;
(2)萌发:
将10-12颗消毒后的种子种脐朝下放置在装有萌发培养基(B5盐类+B5维生素+2%蔗糖+0.3%phytagel,pH 5.8)的120×25mm培养皿中。将培养皿放置在光照培养箱中28℃光照培养4-6天,直到子叶竖起,种皮大部分脱落(28℃,18hr光照,光强140moles/m2/sec);
(3)摇菌:
种子萌发的当天,取-80℃甘油贮存、含有质粒载体的发根农杆菌菌株K599划线于含有相应抗生素的YEP固体平板上,28℃,暗培养2天。2天后,挑取单克隆,置于含有相应抗生素的YEP液体3mL中,28℃,250rpm培养过夜,备用;
(4)侵染和共培养:
用解剖刀从根系上切下发芽的种子,切口在离子叶节大约0.5cm的下胚轴区域。沿着子叶下胚轴垂直剖开种子,并去除子叶上胚轴(幼芽)及轴上的茎/芽。然后用解剖刀蘸取菌液,在子叶节附近制造7-8个切口,将切好的外植体放在装有无菌水润湿的双层滤纸的培养皿中,封上保鲜膜,25℃光照培养5天,然后转到含抑菌剂羧苄青霉素和筛选剂除草剂的发根诱导培养基(MS盐类+B5维生素+3%蔗糖+0.3%phytagel+500mg/L羧卞青霉素+5mg/L除草剂,pH5.8)上25℃暗培养;
(5)继代:
在发根培养基上约10-15天后可见毛状根产生,可将其切下继代到新的发根诱导培养基上,3-4代后不再加抑菌剂,可用于各种分析;
(6)取下大豆发状根,用CTAB法提取大豆发状根基因组DNA;
(7)先用抗性基因Bar.F/R检测转基因率,随后用相应基因的特异引物扩增,回收PCR产物测序,分别统计Spcas9和Sacas9体系对目的基因的编辑情况。
三.汇总并分析数据
通过统计结果可以说明:
(1)splitSpCas9***可运用于大豆基因组编辑,且编辑效率与SpCas9***相近(如下表3所示)
表3
(2)大豆的根毛***中没有检测到SaCas9***介导的基因编辑,且瞬转大豆的根毛长势较差,周期较长,SaCas9***可能对于大豆根毛的生长具有一定的毒性或者抑制作用(如图1所示,数据结果见下表4),
表4
(3)splitSaCas9***可应用于大豆基因组编辑,且效率较高(如下表5、图2C和图2D所示),
表5
检测结果显示,突变类型多样,可造成缺失、增加、替换等多种突变类型,并且可获得纯合突变类型(如图2B所示),另外杂合突变类型达75%(24/32);
(4)SpCas9***与SaCas9***应用于大豆基因组的编辑,从NGG识别位点扩展到NNGRRT识别位点,丰富了CRISPR/Cas***在大豆基因组中的编辑范围,同时也为在今后解决农作物基因组编辑效率的问题上提供新思路。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (3)
1.一种高效率大豆CRISPR/Cas9***的构建方法,其特征在于包括以下步骤:
步骤1)人工合成大豆特异的U6启动子序列、Staphylococcus aureus Cas9序列和Streptococcus pyogenes Cas9序列;
步骤2)将扩增目的片段GmU6,sgRNA,35S,N-SpCas9,C-SpCas9,Bar片段构建于实验室改造的pCAMBIA1300骨架载体上,获得以下载体骨架:
GmU6::sgRNA-35S::SpCas9-35S::Bar
GmU6::sgRNA-35S::SaCas9-35S::Bar
GmU6::sgRNA-35S::N-SpCas9-35S::C-SpCas9-35S::Bar
GmU6::sgRNA-35S::N-SaCas9-35S::C-SaCas9-35S::Bar;
步骤3)从SoyBase中选择3个大豆基因以及2个大豆gma-miRNA,根据选定的基因以及miRNA设计靶位点序列,合成两段带有BsaI酶切后形成的粘性末端互补序列的引物,长度为19bp-21bp;
步骤4)将sgRNA靶位点引物退火后形成的带有粘性末端的核心序列,组装于步骤2)所构建的载体;
步骤5)获得完整的大豆CRISPR/Cas9编辑载体。
2.一种根据权利要求1所述的高效率大豆CRISPR/Cas9***在大豆基因组修饰中的应用。
3.根据权利要求2所述的高效率大豆CRISPR/Cas9***在大豆基因组修饰中的应用方法,其特征在于包括以下步骤:
(1)将权利要求1中获得的大豆CRISPR/Cas9编辑载体转化K599农杆菌;
(2)侵染大豆子叶,培养获得瞬时转化的大豆根毛;
(3)运用CTAB法提取大豆根毛DNA,测序检测编辑效率。
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