CN116478993A - 一种植物cgbe碱基编辑系统及其应用 - Google Patents
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Abstract
一种植物CGBE碱基编辑系统及其应用,涉及基因学领域。所述植物CGBE碱基编辑系统,在失活的Cas9蛋白基因nSpCas9的基础上,加上来自酵母的尿嘧啶糖基化酶基因yUNG,融合大鼠来源的胞嘧啶脱氨酶基因rAPO的突变体APO1‑R33A,以有效地诱导目标C到G碱基转换,减少不必要的C到W(W=A或T)和indel突变水平。利用本发明的基因编辑系统,可以识别PAM序列为NGG的靶点序列,在预定的植物基因组位点的编辑窗口内产生单个C到G的替换,为植物基因组中实施准确的碱基编辑提供了新的方法和思路。
Description
技术领域
本发明涉及生物技术和植物基因工程技术领域。具体而言,本发明涉及一种新型的CGBE碱基编辑器在水稻基因打靶方面的应用,可以提高水稻中的C到G的碱基编辑效率。
背景技术
导致人类遗传病的遗传变异中绝大多数是点突变,因此开发可诱导精准点突变的单碱基编辑工具有极为重要的意义。基因组编辑技术,特别是基于CRISPR/Cas9系统的基因组编辑技术可以通过同源重组(HR)介导的DNA修复途径来实现在基因组位点中引入特定碱基的替换。CRISPR引导的DNA胞嘧啶和腺嘌呤碱基编辑器已经被广泛应用于基因组编辑上,基于CRISPR系统的基因组编辑技术可以通过同源重组(HR)介导的DNA修复途径来实现在基因组位点中引入特定碱基的替换单碱基编辑工具中,目前基于CRISPR的碱基编辑器包括胞嘧啶碱基编辑器(Cytosine Base Editor,CBE)和腺嘌呤碱基编辑器(Adenine BaseEditor,ABE),已被广泛应用于生命科学的各个领域,而且这两类工具的系统性优化和改造已经取得诸多成果,并已被用于多种遗传病的治疗研究。但是,目前这两种碱基编辑器只能产生嘌呤到嘌呤、嘧啶到嘧啶的转换,并不能产生嘌呤到嘧啶的互换。
已经有文献报道了一种糖基化酶碱基编辑器CGBE,它能够在哺乳动物细胞和部分植物中进行C-G编辑。已有团队利用这个编辑器在哺乳动物细胞中的某些基因上实现了>90%的精度(平均96%)和高达70%的效率(平均14%)的编辑,而在植物细胞中在目前的报道中编辑效率只有不到30%,与动物中的编辑效率以及精度都相差甚远。
发明内容
本发明所要解决的技术问题是提供一种能够实现植物中高效的C到G的碱基转化的CGBE碱基编辑工具。
本发明提供一种植物CGBE碱基编辑系统,其中,所述植物CGBE碱基编辑系统包括第一表达盒和第二表达盒;
其中,所述的第一表达盒为向导RNA表达盒,具有式II结构:P1-A-B-T(I);其中,
(a)P1为第1启动子;
(b)A为无或目标位点引导序列sg;
(c)B为sgRNA支架结构序列;
(d)T为终止序列。
优选地,所述第二表达盒为yUNG-GBE融合蛋白表达盒,所述yUNG-GBE融合蛋白具有式II结构:P2-C-D-E-F-G-H-I-J(II);其中,
(a)P2为第2启动子;
(b)C为无或核定位信号序列NLS;
(c)D为APO1-R33A基因序列;
(d)E为任意的连接肽或连接序列;
(e)F为nSpCas9的基因序列;
(f)G为任意的连接肽或连接序列;
(g)H为所述yUNG的基因序列;
(h)I为无或核定位信号序列NLS;
(i)J为终止子,
其中,c和h至多一个为无。
本发明还提供一种植物胞CGBE基编辑系统,所述的yUNG-GBE融合蛋白表达盒和向导RNA表达盒位于同一表达载体上。
本发明还提供一种CGBE碱基编辑载体,所述CGBE碱基编辑载体包括第一表达盒和第二表达盒,或者所述CGBE碱基编辑载体为编辑载体对,所述编辑载体对分别包括第一表达盒和第二表达盒,第一表达盒为序列表中SEQ ID NO:2所示序列,第二表达盒为序列表中SEQ ID NO:1所示序列。
本发明还提供一种所述的CGBE碱基编辑系统的应用,所述应用包括:利用所述的CGBE碱基编辑载体,实现对水稻基因组的碱基编辑,从而获得含有碱基突变的转基因植物或植物部分。
另一方面,本发明还提供一种将所述的基因组编辑载体基因导入水稻细胞的方法。
本发明还提供一种转基因细胞,所述转基因细胞转入有如上所述的表达盒或如上所述的编辑载体。
SEQ ID NO:1如下,与随附的序列表电子版相同
本发明所提供的一种基于yUNG-GBE系统的碱基编辑载体及其应用,可以应用于对水稻或其他单子叶植物的基因组进行编辑,以水稻基因组上片段为靶标,能够在PAM序列下游的第5-7位的碱基进行突变,实现C to G的碱基精准替换。
附图说明
图1为pHUC411-yUNG-GBE载体质粒示意图
图2为yUNG-GBE基因编辑系统产生的碱基编辑效果。
具体实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
实施例1——pHUC411-yUNG-GBE表达载体的构建
所述的yUNG-GBE蛋白包括了APO-R33A变体,nCAS9与yUNG三个部分组成,yUNG-GBE的编码序列如SEQ ID NO:1所示。yUNG-GBE的APO1-R33A与nCAS9,nCAS9与yUNG之间分别插入了连接肽,合成的yUNG-GBE基因两端带上PstI和SacI酶切位点为方便克隆至pHUC411表达载体而设置。将yUNG-GBE合成并连接于PUC57-AMP载体上,形成PUC57-AMP-yUNG-GBE载体,并装载入大肠杆菌XL-blue菌株中。
本发明采用的表达盒包括crRNA表达盒,该crRNA表达盒从5'-3'具有以下4个元件:U3启动子、两个BsaI酶切位点用于对靶标序列进行无缝克隆、sgRNA的骨架结构和转录终止子序列,具体序列见SEQ ID NO:2。合成的crRNA表达盒两端带上Hind III酶切位点为方便克隆至pHUC411表达载体而设置。将crRNA合成并连接于PUC57-crRNA载体上,形成PUC57-AMP-crRNA载体,并装载入大肠杆菌XL-blue菌株中。加粗碱基表示Hind III酶切位点,划线部分碱基为两个BsaI酶切位点相关的序列,构建碱基编辑载体时该序列会被靶标序列代替;黑色底纹为转录终止子序列TTTTTTT;其余为OsU3启动子的序列。
构建载体时,从上面含有PUC57-AMP-yUNG-GBE载体的大肠杆菌XL-blue,用Axygen质粒提取试剂盒中提取质粒,用PstI/SacI酶切,回收yUNG-GBE片段。同时利用PstI/SacI酶对pHUC400进行线性化处理,回收pHUC400,将上述的yUNG-GBE片段和pHUC400片段用T4连接酶(购于TaKaRa公司)进行连接,得到植物表达载体pHUC400-yUNG-GBE。进一步地,通过HindⅢ酶切位点将crRNA表达盒表达框连入pHUC400-yUNG-GBE,得到pHUC411-yUNG-GBE(图1)。
实施例2——利用yUNG-GBE系统对水稻内源基因进行C-G单碱基替换
选择水稻TAC基因(Os09g0529300)中的核苷酸序列AAATGCCGCAAAAGGTGAAAGGG(下划线部分为所述5’-GGG-3’结构的PAM序列),作为打靶位点。合成如下靶标序列,
FP:GGCAAAATCCCGCAAAAGGTGAAA
RP:AAACTTTCACCTTTTGCGGGATTT
斜体部分为载体上的接头序列,将FP和RP退火形成双链。
同时用BsaI酶切pHUC411-yUNG-GBE,通过T4连接酶将靶标双链产物与pHUC411-yUNG-GBE进行连接,形成靶标编辑载体pHUC411-yUNG-GBE-TAC。利用冻融法将这个植物表达载体转入根癌农杆菌(Agrobacterium tumefaciens)EHA105菌株中(安徽省农业科学院水稻研究所保存),然后通过该菌株侵染水稻品种日本晴(Oryza sativa ssp japonicacv.Nipponbare)的愈伤组织,共培养3天后所转化的愈伤组织转移到含有潮霉素的筛选培养中,培养15天后转移到含有潮霉素的分化培养基中再生植株。
采用上述“植物表达载体的构建和农杆菌的转化”过程中转入了重组表达载体的根癌农杆菌,对水稻愈伤组织进行遗传转化。该遗传转化、转化子筛选及转基因植株再生等参照Yongbo Duan(Yongbo Duan,Chenguang Zhai,et al.An efficient and high-throughput protocol for Agrobacterium mediated transformation based onphosphomannose isomerase positive selection in Japonica rice(Oryza sativa L.)[J].Plant Cell Report,2012.DOI 10.1007/s00299-012-1275-3.)等提出的方法。
利用上面构建的载体分别得到转基因植株50株。采取水稻叶片样品,用CTAB法提取DNA。将所得到的基因组DNA样品用于PCR分析。用于扩增靶标附近序列的PCR引物为5’-TGTGATCATAGTGAAGTTAT-3’及5’-AGACACGTCAACTGTAGCTT-3’,产生长度为460bp的片段。将PCR组分首先在95℃保持5分钟,然后进行32个循环:94℃45秒、56℃45秒、72℃45秒,最后在72℃延伸10分钟。将PCR产物测序。所测结果与野生型序列进行比对。结果显示,在pHUC411-yUNG-GBE-TAC这个碱基载体获得的植株中,21棵植株出现了C突变成G,突变效率40.2%(表1),在检测PDS的靶标突变植株中,均在PAM远端的第5位的C突变成G(图2),没有发现其他类型的突变。而且10株出现了只有C突变成G的突变,而不存在C突变成T的情况,纯合突变效率为20.8%。由此可见,本发明的yUNG-GBE单碱基编辑系统能获得较高的C到G的替换,即获得“干净”突变植株效率很高,为水稻育种领域提供了有利的精准改良工具。
表1pHUC411-yUNG-GBE获得的定点突变植株突变类型
按照上述方式进行3次重复实验,所获得的纯净突变效率维持在平均20%±3,具有统计学意义。
Claims (9)
1.一种植物CGBE碱基编辑系统,其特征在于,所述植物CGBE碱基编辑系统包括第一表达盒和第二表达盒;
其中,所述第一表达盒为向导RNA表达盒,具有式I结构:P1-A-B-T(II);其中,
(a)P1为第1启动子;
(b)A为无或目标位点引导序列sg;
(c)B为sgRNA支架结构序列;
(d)T为终止序列。
2.根据权利要求1所述的植物CGBE碱基编辑系统,其特征在于,所述第一表达盒由序列表中SEQ ID NO:2所示的核苷酸序列构成。
3.根据权利要求1所述的植物CGBE碱基编辑系统,其特征在于,所述第二表达盒为yUNG-GBE融合蛋白表达盒,所述yUNG-GBE融合蛋白具有式II结构:P2-C-D-E-F-G-H-I-J(II);其中,
(a)P2为第2启动子;
(b)C为无或核定位信号序列NLS;
(c)D为APO1-R33A基因序列;
(d)E为任意的连接肽或连接序列;
(e)F为nSpCas9的基因序列;
(f)G为任意的连接肽或连接序列;
(g)H为所述yUNG的基因序列;
(h)I为无或核定位信号序列NLS;
(i)J为终止子,
其中,c和h至多一个为无。
4.根据权利要求3所述的植物CGBE碱基编辑系统,所述的yUNG-GBE的DNA切割活性缺失,由序列表中SEQ ID NO:1所示的核苷酸序列构成。
5.根据权利要求3所述的植物胞嘧啶碱基编辑系统,其特征在于,所述的第一表达盒和第二表达盒位于同一表达载体上。
6.根据权利要求3所述的植物CGBE碱基编辑系统,其特征在于,所述的第一表达盒和第二表达盒分别连接在不同表达载体上,两个表达载体置于同一遗传转化系统中。
7.一种CGBE碱基编辑载体,所述CGBE碱基编辑载体包括第一表达盒和第二表达盒,或者所述CGBE碱基编辑载体为编辑载体对,所述编辑载体对分别包括第一表达盒和第二表达盒,第一表达盒为序列表中SEQ ID NO:2所示序列,第二表达盒为序列表中SEQ ID NO:1所示序列。
8.一种权利要求1-4中任意一项所述的植物CGBE碱基编辑系统的应用,其特征在于,所述应用包括:利用所述植物CGBE碱基编辑系统,对水稻基因组中预定位置的C碱基进行碱基编辑,使其突变至G碱基,从而获得含有碱基突变的转基因植物或植物部分。
9.一种将权利要求1中所述的植物CGBE碱基编辑系统导入水稻细胞的方法,其特征在于,所述方法包括:
(1)构建pHUC411-yUNG-GBE表达载体,所述pHUC411-yUNG-GBE表达载体中包含序列表中SEQ ID NO:1所示的核苷酸序列;
(2)构建crRNA表达盒,并在所述crRNA表达盒两端带上Hind III酶切位点,并构建含有crRNA表达盒的第二载体;
(3)将所构建的pHUC411-yUNG-GBE表达载体和第二载体转入根癌农杆菌中侵染水稻愈伤组织,并再生相应植株。
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