CN106434737A - 基于CRISPR/Cas9技术的单子叶植物基因敲除载体及其应用 - Google Patents

基于CRISPR/Cas9技术的单子叶植物基因敲除载体及其应用 Download PDF

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CN106434737A
CN106434737A CN201610197562.9A CN201610197562A CN106434737A CN 106434737 A CN106434737 A CN 106434737A CN 201610197562 A CN201610197562 A CN 201610197562A CN 106434737 A CN106434737 A CN 106434737A
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cas9
crispr
sgrna
bspq
gene knockout
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张业胜
张如
郝志强
李臻
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Inner Mongolia Zhongke Positive Biological Science And Technology Co Ltd
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Abstract

本发明公开了一种基于CRISPR/Cas9技术的植物基因敲除载体及其应用，该植物基因敲除载体为单子叶植物CRISPR/Cas9敲除载体pCAMBIA1300DM‑OsU3(BspQ I)‑Cas9，其中含有BspQ I的插入位点。本发明的有益效果在于：由BspQ I插入位点组成的sgRNA‑OsU3结构序列与2x35s‑hSpCas9‑ter序列共同构建的CRISPR/Cas9植物敲除的双元载体pCAMBIA1300DM‑OsU3(BspQ I)‑Cas9，能够通过一步酶切连接的方法来实现高效、快速构建单子叶植物基因敲除载体的目的；本发明提供的CRISPR/Cas9敲除载体pCAMBIA1300DM‑OsU3(BspQ I)‑Cas9，具有打靶效率高、脱靶效率低的特点，能够应用于植物靶基因的敲除或者以试剂盒的形式应用于植物基因编辑。

Description

基于CRISPR/Cas9技术的单子叶植物基因敲除载体及其应用
技术领域：
本发明属于生物技术领域，具体涉及基于CRISPR/Cas9技术的单子叶植物基因敲除载体及其应用。
背景技术
II型CRISPR/Cas9系统是来自链球菌属的一种获得性免疫系统，能够抵御外源基因的入侵。经过人工修饰，这种系统在动植物中得到了广泛应用。它主要由CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)和特异的Cas9蛋白组成。CRISPR是一成簇的规律间隔的短回文DNA重复序列，它由一系列短的高度保守的正向重复序列和长度相似的序列间隔排列组成。Cas9蛋白是一种由1409个氨基酸组成的多结构域蛋白，含有两个核酸酶结构域RuvC-like和HNH。CRISPR/Cas9系统于2013年被Seung等人(Seung Woo Cho et al.,2013)首次成功运用于真核细胞的基因组编辑。基于CRISPR/Cas9的基因组编辑体系包括2部分：sgRNA及酶Cas9。这种系统能够特异的识别毗邻PAM(NGG)基序的靶序列，并在其上游3nt处进行切割，同时RuvC-like结构域在PAM区上游3～8nt处进行切割，形成DSB(double strains break),随后机体通过自身的修复机制如同源重组、非同源重组来修复损伤的DNA,进而产生Indel(包括缺失和插入)的现象。
目前应用于植物上的CRISPR/Cas9载体构建体系并不多，有基于基因枪转化的载体构建体系和基于农杆菌转化的载体构建体系。在目前普遍使用的植物农杆菌转化方面，基于CRISPR/Cas9技术的植物基因敲除载体体系构建过程比较繁琐，需要多步才能将各个部分整合到双元载体上。已报道的CRISPR/Cas9体系，有的体系是借助限制性内切酶BbsI先把sgRNA插入pATU6-26SK/pOsU6SK载体,再通过限制性内切酶KpnI和SalI酶切得到AtU6-sgRNA片段或通过KpnI和HindIII双酶切获得OsU6-sgRNA片段，同时将酶切过的SalI-Cas9-EcoRI或HindIII-Cas9-EcoRI片段与已用KpnI和EcoRI酶切的线性化的质粒pCAMBIA1300连接构建双元载体，这种系统在构建过程中比较复杂而且周期长(ZY Feng,JK Zhu et al.,2013)；另外有利用合成的方法直接合成启动子和sgRNA单元，再构建到双元载体上，这种体系成本高，周期长(Wenzhi Jiang et al.,2013)。而在基因枪转化方面中，已报道的体系都是将pU6-gRNA或pU3-gRNA载体与Cas9载体共同包裹在金粉中打入植物组织中，这种费用比较高。以上这些体系，要完成一个植物基因CRISPR/Cas9敲除载体的构建，要么成本高昂，要么过程繁琐，且不能满足高通量植物基因CRISPR/Cas9敲除载体构建的需求。
还有一种体系是将启动子、sgRNA scaffold和Cas9片段整合到一个双元载体，并选用BsaI作为插入位点，但是这种体系的缺陷在于，在两个BsaI之间存在SpR(spectinomycin resistance gene壮观霉素抗性基因)片段，在构建过程中需要Bsa I酶切回收线性化载体，再连接target site(HL Xing,L Dong et al.,2014)。
而本发明是将水稻OsU3启动子及sgRNA元件与35S启动子启动的hSpCas9元件直接整合在已经突变掉两个BspQ I识别位点的双元载体pCAMBIA1300DM上，并在sgRANscaffold元件中选用BspQ I作为靶序列的插入位点，且两个BspQ I只间隔7bp碱基，只需要通过酶切纯化获取线性化载体，再一步连接的方法插入target oligos，实现简便、快速和高效的构建CRISPR/Cas9基因敲除载体。
发明内容
本发明的一个目的是提供一种基于CRISPR/Cas9技术的单子叶植物基因敲除载体pCambin1300DM-OsU3-Cas9，其具有以下特征：
(1)骨架载体为双元载体pCAMBIA1300DM，所述双元载体pCAMBIA1300DM是pCAMBIA1300中原有的2个BspQI酶切位点gaaGAgc和gcTcttc分别被突变成gaaCTgc和gcActtc的载体；
(2)在所述双元载体pCAMBIA1300DM上，同时连入了sgRNA元件和Cas9元件；
(3)所述sgRNA元件由sgRNA启动子、sgRNA-scaffold和靶点插入酶切位点组成；
(4)所述sgRNA启动子为OsU3启动子；
(5)所述靶点插入酶切位点为BspQ I，酶切位点信息为：
BspQ I:GGCAGAAGAGCAACACAAGCTCTTCA(SEQ ID NO：1)(6)所述Cas9元件为2x35S-Cas9-ter，其中2x35S为串联的2个35S启动子，ter为终止子(terminator)，Cas9为编码基因Cas9的全长CDS序列并且两端加有核定位的信号NLS(Nuclear localization signal)，N端加有Flag标签。
本发明是将sgRNA结构单元与Cas9基因共同整合到骨架载体pCAMBIA1300DM上的敲除载体pCAMBIA1300DM-OsU3-Cas9，同时sgRNA结构单元中有BspQI的插入位点，其序列如下：
BspQ I:GGCAGAAGAGCAACACAAGCTCTTCA(SEQ ID NO：1)
本发明使用的启动子是OsU3启动子，在植物中启动sgRNA scaffold结构的启动子属于Pol III promoters，包括U3和U6的启动子，而OsU3是单子叶植物中启动效率较高的启动子；同时选用的限制性酶的插入位点也是一类特征的酶—Type IIs限制性内切酶，可选用的酶有AarI、BspQI、BbsI/Bpil、BsmBI/Esp3I、BfuAI/BveI和BsaI/Eco31I，而在本发明中选用BspQI的插入位点。
本发明使用pCAMBIA1300DM(原pCAMBIA1300的载体序列上有2处BspQ I酶切位点，为能使BspQ I作为靶位点插入使用的酶切位点，对传统的pCAMBIA1300上原有的2处BspQ I酶切位点做了突变，使其成为本身没有BspQ I酶切位点的双元载体，命名为pCAMBIA1300DM)作为骨架载体，将2x35s-Cas9-ter(2x35s：2个串联在一起的35s启动子；Cas9:密码子优化的Cas9酶的CDS序列；ter:终止子序列)及sgRNA元件(使用水稻的OsU3启动子启动)连入骨架载体；同时将BspQ I的酶切位点连入sgRNA元件的靶位点插入处(sgRNA启动子与sgRNA scaffold之间)，构建出适用于单子叶植物基因的敲除载体pCAMBIA1300DM-OsU3(BspQ I)-Cas9。
2x35s-Cas9-ter的序列(SEQ ID NO：2)：
CGGTATCGATAAGCTTGCATGCCTGCAGGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTGATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACCTCGACCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATACTCGAGATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAAGGATCCTGATTGATCGATAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGG
sgRNAscaffold的序列(SEQ ID NO：3)：
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTT
本发明基于CRISPR/Cas9技术的植物基因敲除载体能够应用于植物靶基因的敲除，同时也能够以试剂盒的形式应用于植物基因编辑。
本发明的有益效果在于：
1、由BspQ I插入位点组成的sgRNA-OsU3结构序列与2x35s-hSpCas9-ter序列共同构建的CRISPR/Cas9植物敲除的双元载体pCAMBIA1300DM-OsU3(BspQ I)-Cas9，能够通过一步酶切连接的方法来实现高效、快速构建单子叶植物基因敲除载体的目的。
2、本发明提供的CRISPR/Cas9敲除载体pCAMBIA1300DM-OsU3(BspQ I)-Cas9，具有打靶效率高、脱靶效率低的特点，本发明载体的打靶效率高于10％，优选达到60％，特别适合应用于植物靶基因的敲除或者以试剂盒的形式运用到植物基因编辑中。
附图说明
图1为单子叶基因敲除载体结构图—pCAMBIA1300DM-OsU3(BspQ I)-Cas9。
图2为载体pCAMBIA1300DM的结构图。
图3为Gn1a基因的测序结果与峰图。
图4为OsDEP1基因的测序结果与峰图。
具体实施方案：
一.单子叶基因敲除载体构建：
1.将pOsU3-gRNA载体(中科院遗传与发育研究所高彩霞实验室馈赠)用Aar I酶切成线性化载体，并经CIP(碱性磷酸酶)反应后纯化。
2.将合成的含有BspQ I的酶切位点的oligos序列进行退火磷酸化反应。
BspQ I-oligo1:GGCAGAAGAGCAACACAAGCTCTTCA(SEQ ID NO：5)
BspQ I-oligo2:AAACTGAAGAGCTTGTGTTGCTCTTCT(SEQ ID NO：6)
3.将退火磷酸化后的BspQ I插入序列与pOsU3-gRNA线性化载体进行T4连接，将BspQ I插入序列连入载体中。挑选阳性克隆的质粒(Aar I酶切不开的质粒)送测序，所测得正确的OsU3-sgRNA序列的载体标记为pOsU3-gRNA-B，其中OsU3(BspQ I)-sgRNAscaffold结构序列(画下划线的为BspQ I的识别序列)为(SEQ ID NO：4)：
AAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTCGGAAACCACGTGATGTGAAGAAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGATTTAAAAGAGTTGTGCAGATGATCCGTGGCAGAAG AGCAACACAAGCTCTTCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCCACATAATCTCTAGAGGATC.
4.设计含有AscI酶切序列的AscI-OsU3-F/R引物并从pOsU3-gRNA-B载体上扩增OsU3-sgRNAscaffold片段，
AscI-OsU3-F：5’-TTGGCGCGCCAGGAATCTTTAAACATACGAAC(SEQID NO：7)；
AscI-OsU3-R：5’-TTGGCGCCGATCCTCTAGAGATTATGTGG(SEQ ID NO：8)。
PCR反应条件为95℃，30s；65℃，30s；72℃，40s；35cycles。
5.胶回收OsU3-sgRNA scaffold的片段，连入T载体PMD19-T进行T/A克隆，并挑取阳性克隆送测序，获得含有OsU3-sgRNA scaffold结构的PMD19-T载体。
6.酶切获得AscI-OsU3-sgRNAscaffold片段：直接用AscI限制性内切酶从pMD19-T质粒上酶切获得AscI-OsU3-sgRNAscaffold片段。并与已用AscI酶切线性化的pCAMBIA1300DM进行T4连接，构建pCAMBIA1300DM-OsU3质粒，构建成功后将质粒送去测序(测序引物OsU3-F:CCCAAGCTTAGGAATCTTTAAACATACGAAC(SEQ ID NO：9))。
7.从2X35s-hSpCas9质粒(中科院上海生命科学研究院朱健康实验室馈赠)上用EcoRI和SalI酶酶切得到2x35s-hSpCas9-ter片段，并进行胶回收。同时用EcoRI和SalI酶切pCAMBIA1300DM-OsU3(BspQ I位点)质粒，获得线性化质粒。
6.将2x35s-hSpCas9-ter片段连入pCAMBIA1300DM-OsU3线性化质粒中，经转化、挑克隆、酶切检测和测序，获得正确的pCAMBIA1300DM-OsU3-Cas9质粒。
实施例：
一、水稻基因Gn1a敲除载体的构建
1.根据水稻基因Gn1a序列和sgRNA设计工具设计特意的靶序列，序列如下：
Gn1a-oligo1:GGCAGCAGTACCTGCCTTACTA(SEQ ID NO：10)
Gn1a-oligo2:AAACTAGTAAGGCAGGTACTGC(SEQ ID NO：11)
2.将设计的targetoligos按如下条件进行退火磷酸化过程：
在PCR管中配制反应体系：
在PCR仪上按以下程序完成反应过程：
①37℃30min
②95℃ 5min
③60个循环：
95℃—1.3℃/cycle 45s
④4℃保存
3.用BspQ I酶酶切pCAMBIA1300DM-OsU3-Cas9质粒，获取线性化载体，并用CIP酶去做磷酸化处理，以免自连。
4.将退火磷酸化产物稀释100倍，取2μl与线性化载体进行T4连接：
在PCR管中配制连接体系：
16℃连接0.5～1h，连接产物转入大肠杆菌DH5a感受态细胞中并筛选阳性克隆2～4个送去测序。成功插入靶位点的测序结果为：
AGTTGTGCAGATGATCCGTGGCAGCAGTACCTGCCTTACTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTT(SEQ ID NO：12)
表明水稻基因Gn1a的CRIPSR/Cas9敲除载体构建成功。
5.将其敲除载体转至农杆菌中，并完成整个组培过程，获得转基因阳性幼苗，并做检测验证。
6.目的片段PCR产物的测序结果表明在53株转基因水稻中有26株出现突变现象，这表明Gn1a基因的打靶效率为49％，见附图3。
二、水稻DEP1基因的CRISPR/Cas9敲除载体的构建
1.根据水稻OsDEP1基因序列和sgRNA设计工具设计特意性的靶序列，序列如下：
OsDEP1-oligo1:GGCAGCTGCGGTTGCAACGGCTG(SEQ ID NO：13)
OsDEP1-oligo2:AAACCAGCCGTTGCAACCGCAGC(SEQ ID NO：14)
2.其他步骤与OsPDS基因的载体构建过程一样。
3.OsDEP1转基因阳性植株的检测：利用OsDEP1-F/R引物
OsDEP1-F:5’-GCTGCTCATGCTGTAAACCT-3’(SEQ ID NO：15)
OsDEP1-R:5’-CGTGGGCATCGACAACCCTC-3’(SEQ ID NO：16)
进行目的片段PCR扩增，并将PCR产物进行测序，PCR反应条件为95℃，30s；55℃，30s；72℃，40s；35cycles。
4.分析测序结果:48株转基因水稻中有19株发生了不同程度的突变，缺失最多的碱基数达30bp，突变频率为39％，见附图4。
从这些实施例可知，本发明所构建的单子叶敲除载体pCAMBIA1300DM(BspQ I)-OsU3-Cas9具有简便、快速、高效的优点，特别是具有较高的打靶效率和极低的脱靶率。
所属领域的普通技术人员应当理解：以上任何实施例的讨论仅为示例性的，并非旨在暗示本公开的范围(包括权利要求)被限于这些例子；在本发明的思路下，以上实施例或者不同实施例中的技术特征之间也可以进行组合，步骤可以以任意顺序实现，并存在如上所述的本发明的不同方面的许多其它变化，为了简明它们没有在细节中提供。因此，凡在本发明的精神和原则之内，所做的任何省略、修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (7)

1.一种基于CRISPR/Cas9技术的植物基因敲除载体，其特征在于该植物基因敲除载体为单子叶植物CRISPR/Cas9敲除载体pCAMBIA1300DM-OsU3(BspQ I)-Cas9，其中含有BspQ I的插入位点。
2.如权利要求1所述的基于CRISPR/Cas9技术的植物基因敲除载体，其特征在于，所述植物基因敲除载体具有以下特征：
(1)骨架载体为双元载体pCAMBIA1300DM，所述双元载体pCAMBIA1300DM是pCAMBIA1300中原有的2个BspQ I酶切位点gaaGAgc和gcTcttc分别被突变成gaaCTgc和gcActtc的载体；
(2)在所述双元载体pCAMBIA1300DM上，同时连入了sgRNA元件和Cas9元件；
(3)所述sgRNA元件由sgRNA启动子、sgRNA-scaffold和靶点插入酶切位点组成；
(4)所述sgRNA启动子为OsU3启动子；
(5)所述靶点插入酶切位点为BspQI,酶切位点信息为：
BspQI:GGCAGAAGAGCAACACAAGCTCTCA(SEQ ID NO：1)(6)所述Cas9元件为2x35S-Cas9-ter，其中2x35S为串联的2个35S启动子，ter为终止子，Cas9为编码基因Cas9的全长CDS序列并且两端加有核定位的信号NLS，N端加有Flag标签。
3.如权利要求2所述的基于CRISPR/Cas9技术的植物基因敲除载体，其特征在于，所述2x35S-Cas9-ter的序列为SEQ ID NO：2。
4.如权利要求2所述的基于CRISPR/Cas9技术的植物基因敲除载体，其特征在于，所述sgRNA-scaffold的序列为SEQ ID NO：3。
5.如权利要求2所述的基于CRISPR/Cas9技术的植物基因敲除载体，其特征在于，所述sgRNA元件为OsU3(BspQ I)-sgRNA，其序列为SEQ ID NO：4。
6.如权利要求1-5中任一项所述的CRISPR/Cas9植物基因敲除载体在单子叶植物的基因敲除中的应用。
7.如权利要求1-5中任一项所述的CRISPR/Cas9植物基因敲除载体作为试剂盒用于植物基因编辑的应用。
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Publication number Priority date Publication date Assignee Title
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
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US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
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US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
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US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103343120A (zh) * 2013-07-04 2013-10-09 中国科学院遗传与发育生物学研究所 一种小麦基因组定点改造方法
CN103382468A (zh) * 2013-07-04 2013-11-06 中国科学院遗传与发育生物学研究所 一种水稻基因组定点改造方法
CN103667338A (zh) * 2013-11-28 2014-03-26 中国科学院遗传与发育生物学研究所 一种玉米基因组定点改造方法
CN103911376A (zh) * 2014-04-03 2014-07-09 南京大学 CRISPR-Cas9靶向敲除乙肝病毒cccDNA及其特异性sgRNA
CN104004778A (zh) * 2014-06-06 2014-08-27 重庆高圣生物医药有限责任公司 含有CRISPR/Cas9系统的靶向敲除载体及其腺病毒和应用
CN104232669A (zh) * 2014-08-25 2014-12-24 中国水产科学研究院黑龙江水产研究所 一种采用基因敲除法构建基于鱼类CRISPR/Cas9系统的载体及其构建方法
CN104293828A (zh) * 2013-07-16 2015-01-21 中国科学院上海生命科学研究院 植物基因组定点修饰方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103343120A (zh) * 2013-07-04 2013-10-09 中国科学院遗传与发育生物学研究所 一种小麦基因组定点改造方法
CN103382468A (zh) * 2013-07-04 2013-11-06 中国科学院遗传与发育生物学研究所 一种水稻基因组定点改造方法
CN104293828A (zh) * 2013-07-16 2015-01-21 中国科学院上海生命科学研究院 植物基因组定点修饰方法
CN103667338A (zh) * 2013-11-28 2014-03-26 中国科学院遗传与发育生物学研究所 一种玉米基因组定点改造方法
CN103911376A (zh) * 2014-04-03 2014-07-09 南京大学 CRISPR-Cas9靶向敲除乙肝病毒cccDNA及其特异性sgRNA
CN104004778A (zh) * 2014-06-06 2014-08-27 重庆高圣生物医药有限责任公司 含有CRISPR/Cas9系统的靶向敲除载体及其腺病毒和应用
CN104232669A (zh) * 2014-08-25 2014-12-24 中国水产科学研究院黑龙江水产研究所 一种采用基因敲除法构建基于鱼类CRISPR/Cas9系统的载体及其构建方法

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Application publication date: 20170222