CN106167810A - 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

Info

Publication number
CN106167810A
CN106167810A CN201610197563.3A CN201610197563A CN106167810A CN 106167810 A CN106167810 A CN 106167810A CN 201610197563 A CN201610197563 A CN 201610197563A CN 106167810 A CN106167810 A CN 106167810A
Authority
CN
China
Prior art keywords
cas9
crispr
knockout carrier
sgrna
osu3
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610197563.3A
Other languages
English (en)
Inventor
张业胜
张如
郝志强
李臻
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inner Mongolia Zhongke Positive Biological Science And Technology Co Ltd
Original Assignee
Inner Mongolia Zhongke Positive Biological Science And Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Inner Mongolia Zhongke Positive Biological Science And Technology Co Ltd filed Critical Inner Mongolia Zhongke Positive Biological Science And Technology Co Ltd
Publication of CN106167810A publication Critical patent/CN106167810A/zh
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明公开了一种基于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pCambin1300‑OsU3‑Cas9，含有Aar I插入位点。本发明的有益效果在于：由Aar I插入位点组成的sgRNA‑OsU3结构序列与2x35s‑Cas9‑ter序列共同构建的CRISPR/Cas9植物敲除的双元载体pCambin1300‑OsU3‑2x35s‑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pCambin1300‑OsU3‑2x35s‑Cas9，具有打靶效率高、脱靶效率极低的特点，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双链断裂),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Bsa I作为插入位点，但是这种体系的缺陷在于，在两个Bsa I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 sites(靶位)(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Cas9元件直接整合在双元载体pCAMBIA1300上，并在sgRAN scaffold元件中选用Aar I作为靶序列的插入位点，且两个Aar 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，从而实现简便、快速和高效的构建CRISPR/Cas9基因敲除载体。
发明内容
本发明的一个目的在于提供一种基于CRISPR/Cas9技术的单子叶植物基因敲除载体pCAMBIA1300-OsU3-2x35s-Cas9，该载体具有以下特征：
(1)骨架载体为双元载体pCAMBIA1300；
(2)在所述双元载体上，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及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Aar I,酶切位点信息为：
Aar I:CTTGGCTCGCAGGTGAACACAACACCTGCACAC(SEQ ID NO：1)
(6)所述Cas9元件为2x35S-Cas9-ter，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pCAMBIA1300上的敲除载体pCAMBIA1300-OsU3-2x35s-Cas9，同时sgRNA结构单元中有Aar I的插入位点，其序列如下：
Aar I:CTTGGCTCGCAGGTGAACACAACACCTGCACAC(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Aar I、BspQ I、Bbs I/Bpi l、BsmB I/Esp3 I、BfuA I/Be I和Bsa I/Eco31 I，而在本发明中选用Aar I的插入位点(其他位点在pCAMBIA1300载体上都存在)。
本发明使用pCAMBIA1300作为骨架载体，将2x35s-Cas9-ter(2x35s：2个串联在一起的35S启动子；cas9:cas9酶的CDS序列；ter:终止子序列)及sgRNA元件(单子叶植物使用水稻的OsU3启动子启动)连入骨架载体；同时将Aar 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敲除载体pCAMBIA1300-OsU3(Aar I)-2x35s-Cas9。
2x35s-Cas9-ter的序列(SEQ ID NO：2)：
CGGTATCGATAAGCTTGCATGCCTGCAGGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTGATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACCTCGACCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATACTCGAGATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAAGGATCCTGATTGATCGATAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGG
sgRNA scaffold的序列为(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到植物靶基因的敲除中，同时也能够以试剂盒的形式运用到植物基因编辑中。
本发明的有益效果在于：
1、由Aar I插入位点组成的sgRNA-OsU3结构序列与2x35s-Cas9-ter序列共同构建的CRISPR/Cas9植物敲除的双元载体pCAMBIA1300-OsU3-2x35s-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pCAMBIA1300-OsU3-2x35s-Cas9，具有打靶效率高、脱靶效率极低的特点，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—pCAMBIA1300-OsU3(Aar I)-Cas9。
图2为敲除基因OsPDS的水稻表型及检测的酶切和测序结果图。
图3为敲除基因Gn1a的测序结果和测序峰图
具体实施方案：
一.单子叶基因敲除载体构建：
1.设计含有Hind III酶切序列的Hind III-OsU3-F/R引物并扩增质粒pOsU3-gRNA中的OsU3(Aar I插入位点)(中科院遗传与发育研究所高彩霞实验室馈赠)片段，
Hind III-OsU3-F：5’-CCCAAGCTTAGGAATCTTTAAACATACGAAC-3’(SEQ ID NO：5)；
Hind III-OsU3-R：5’-CAAGCTTGATCCTCTAGAGATTATGTGG-3’(SEQ ID NO：6)
PCR反应条件95℃，30s；56℃，30s；72℃，40s；35cycles。
2.胶回收OsU3片段，连入T载体PMD19-T进行T/A克隆，并挑选阳性克隆送测序，所测得的OsU3(Aar I)-sgRNA结构序列(画下划线的为Aar I的识别序列)为(SEQ ID NO：4)：
AAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTCGGAAACCACGTGATGTGAAGAAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGATTTAAAAGAGTTGTGCAGATGATCCGTGGCAGCTCGCAGGTGAACACAACACCTGCACACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCCACATAATCTCTAGAGGATC
3.酶切获得Hind III-OsU3片段：直接用Hind III限制性内切酶从pMD19-T质粒上酶切获得Hind III-OsU3片段。并与已用Hind III酶切线性化的pCAMBIA1300进行T4连接，构建pCAMBIA1300-OsU3质粒，构建成功后将质粒送去测序，其中测序引物OsU3-F的序列为:CCCAAGCTTAGGAATCTTTAAACATACGAAC(SEQ ID NO：5)。
4.从2X35s-Cas9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Sal I酶酶切得到2X35s-Cas9-ter片段，并进行胶回收。同时用EcoR I和Sal I酶切pCAMBIA1300-OsU3质粒，获得线性化质粒。
5.将2X35s-Cas9-ter片段连入pCAMBIA1300-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pCAMBIA300-OsU3(Aar I)-Cas9质粒。
实施例1：水稻白化基因的敲除与验证
一、水稻白化基因OsPDS敲除载体的构建
1.根据水稻白化基因OsPDS序列和sgRNA设计工具设计特意的靶序列，序列如下：
OsPDS-oligo1:GGCAGTTGGTCTTTGCTCCTGCAG(SEQ ID NO：7)
OsPDS-oligo2:AAACCTGCAGGAGCAAAGACCAAC(SEQ ID NO：8)
2.将设计的target oligos按如下条件进行退化磷酸化过程：
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：
3.用Aar I酶酶切pCAMBIA1300-OsU3(Aar I)-Cas9质粒，获取线性化载体，并用CIP酶做去磷酸化处理，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：
GGCAGTTGGTCTTTGCTCCTGCAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTT
表明水稻白化基因OsPDS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，做检测验证。
6.酶切试验和目的片段PCR产物的测序结果都表明13％的单株中产生了缺失现象，同时植株中出现白化苗表型，见附图2。
实施例2：水稻Gn1a基因的敲除与验证
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-2-oligo1:GGCAGCGGCCAGGCCTTCCGCCA(SEQ ID NO：9)
Gn1a-2-oligo2:AAACTGGCGGAAGGCCTGGCCGC(SEQ ID NO：10)
2.按照实施例1中的步骤成功构建Gn1a的敲除载体。
3.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，做检测验证。
4.Gn1a转基因阳性植株的检测：利用Gn1a-F/R引物
Gn1a-F:5’-GATTGATTGATTGATAATGAAGC-3’(SEQ ID NO：11)；
Gn1a-R:5’-CCTATACCTTAATTACCTC-3’(SEQ ID NO：12)
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。
5.目的片段的PCR产物测序结果：在14株转基因水稻中有5株存在突变现象，即突变频率为35％，见附图3。
从这些实施例可知，本发明所构建的单子叶敲除载体pCAMBIA1300(Aar 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pCAMBIA1300-OsU3(Aar I)-Cas9，其中含有Aar 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pCambia1300；
(2)在所述双元载体上，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及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Aar I，酶切位点信息为：
Aar I:CTTGGCTCGCAGGTGAACACAACACCTGCACAC(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(Aar 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。
CN201610197563.3A 2015-04-03 2016-03-31 基于CRISPR/Cas9技术的单子叶植物基因敲除载体及其应用 Pending CN106167810A (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510156057 2015-04-03
CN2015101560575 2015-04-03

Publications (1)

Publication Number Publication Date
CN106167810A true CN106167810A (zh) 2016-11-30

Family

ID=57359299

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201610197562.9A Pending CN106434737A (zh) 2015-04-03 2016-03-31 基于CRISPR/Cas9技术的单子叶植物基因敲除载体及其应用
CN201610197563.3A Pending CN106167810A (zh) 2015-04-03 2016-03-31 基于CRISPR/Cas9技术的单子叶植物基因敲除载体及其应用

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN201610197562.9A Pending CN106434737A (zh) 2015-04-03 2016-03-31 基于CRISPR/Cas9技术的单子叶植物基因敲除载体及其应用

Country Status (1)

Country Link
CN (2) CN106434737A (zh)

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107254485A (zh) * 2017-05-08 2017-10-17 南京农业大学 一种能够快速构建植物基因定点敲除载体的新反应体系
CN107338265A (zh) * 2017-07-21 2017-11-10 中国科学院遗传与发育生物学研究所 一种基因编辑系统及应用其对植物基因组进行编辑的方法
CN107893086A (zh) * 2017-10-24 2018-04-10 中国科学院武汉植物园 快速构建配对sgRNA的Cas9双元表达载体文库的方法
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
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 (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110256548A (zh) * 2019-07-04 2019-09-20 中国农业科学院生物技术研究所 具有调控植物开花期功能的ZmELF3.1蛋白及其功能缺失突变体和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103343120B (zh) * 2013-07-04 2015-03-04 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
CN103667338B (zh) * 2013-11-28 2016-01-27 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
CN103911376B (zh) * 2014-04-03 2017-02-15 黄行许 CRISPR‑Cas9靶向敲除乙肝病毒cccDNA及其特异性sgRNA
CN104004778B (zh) * 2014-06-06 2016-03-02 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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HUI-LI XING ET AL.: "A CRISPR/Cas9 toolkit for multiplex genome editing in plants", 《BMC PLANT BIOLOGY》 *
QIWEI SHAN ET AL.: "Genome editing in rice and wheat using the CRISPR/Cas system", 《NATURE PROTOCOLS》 *

Cited By (45)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US12006520B2 (en) 2011-07-22 2024-06-11 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11999947B2 (en) 2016-08-03 2024-06-04 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
CN107254485A (zh) * 2017-05-08 2017-10-17 南京农业大学 一种能够快速构建植物基因定点敲除载体的新反应体系
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
CN107338265A (zh) * 2017-07-21 2017-11-10 中国科学院遗传与发育生物学研究所 一种基因编辑系统及应用其对植物基因组进行编辑的方法
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
CN107893086A (zh) * 2017-10-24 2018-04-10 中国科学院武汉植物园 快速构建配对sgRNA的Cas9双元表达载体文库的方法
CN107893086B (zh) * 2017-10-24 2021-09-03 中国科学院武汉植物园 快速构建配对sgRNA的Cas9双元表达载体文库的方法
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
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

Also Published As

Publication number Publication date
CN106434737A (zh) 2017-02-22

Similar Documents

Publication Publication Date Title
CN106167810A (zh) 基于CRISPR/Cas9技术的单子叶植物基因敲除载体及其应用
US11584936B2 (en) Targeted viral-mediated plant genome editing using CRISPR /Cas9
CN105132451B (zh) 一种CRISPR/Cas9单一转录单元定向修饰骨架载体及其应用
JP2021151275A (ja) マーカーフリーゲノム改変のための方法および組成物
CN107027313B (zh) 用于多元rna引导的基因组编辑和其它rna技术的方法和组合物
CN108130342B (zh) 基于Cpf1的植物基因组定点编辑方法
CN103382468B (zh) 一种水稻基因组定点改造方法
Hancock et al. The rice miniature inverted repeat transposable element mPing is an effective insertional mutagen in soybean
Lee et al. CRISPR/Cas9-mediated targeted T-DNA integration in rice
US20200190492A1 (en) Novel anti-crispr polynucleotides and polypeptides and methods of use
CA2994883A1 (en) Method for obtaining glyphosate-resistant rice by site-directed nucleotide substitution
CN107236737A (zh) 特异靶向拟南芥ILK2基因的sgRNA序列及其应用
CN107722125B (zh) 一种人工转录激活因子dCas9-TV及其编码基因与应用
CN107365793A (zh) 一种适用于植物的大规模基因组编辑的方法
WO2019122394A2 (en) Cpf1 based transcription regulation systems in plants
WO2018183878A1 (en) Expression modulating elements and use thereof
CN105112435A (zh) 植物多基因敲除载体的构建及应用
EP3737691A1 (en) Optimized plant crispr/cpf1 systems
CN115315516B (zh) 一种提高植物遗传转化和基因编辑效率的方法
CN106609282A (zh) 一种用于植物基因组定点碱基替换的载体
CN116391038A (zh) 用于改善基因组编辑的工程化Cas内切核酸酶变体
CN103013954B (zh) 水稻基因badh2的定点敲除系统及其应用
CN111902541A (zh) 增加细胞中感兴趣的核酸分子表达水平的方法
Thieme et al. Transposable elements as tool for crop improvement
CN105112440A (zh) 一种与重组酶介导的体内基因叠加相兼容的体外基因叠加技术及其应用

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20161130