Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles.
Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles.
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DOI：
10.1007/s00425-014-2180-5
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发表时间：
2015-01
期刊：
影响因子：
4.3
通讯作者：
Coupland G
中科院分区：
文献类型：
--
作者：
Hyun Y;Kim J;Cho SW;Choi Y;Kim JS;Coupland G
利用CRISPR/Cas系统的Rgen进行组织定向突变，在拟南芥中产生可遗传的突变。高等植物定点基因组工程在基础研究和分子育种方面具有巨大的潜力。在这里，我们描述了一种对拟南芥核基因组进行定点突变的方法，该方法使用来自细菌簇状规则间隔短回文重复序列(CRISPR)-Cas9(CRISPR相关)蛋白系统的RNA引导的内切酶(Rgen)有效地产生可遗传突变。为了在胚胎发生和整个植物生命周期的增殖组织中诱导突变，U6SnRNA和INCURVATA2启动子分别表达了单引导RNA(SgRNA)和Cas9DNA内切酶。通过农杆菌介导法将针对开花基因T(FT)和鳞状病毒启动子结合蛋白类4基因的编码抗原的T-DNA导入T1转化子，在目标基因座上观察到了体细胞突变。在FT-rgen的结果中，T1植株通常表现为开花晚，这表明在两条染色体上都存在着FT基因突变的大型体细胞区段。DNA测序分析估计，大约90%的独立染色体DNA片段在一株表现开花晚的T1植株的分析组织中携带突变。在T2植株中检测到的最常见的体细胞多态表现出较高的遗传率，并且也观察到了较少的多态遗传。结果是，在接下来的T2和T3世代中，恢复了具有新的、可遗传的FT零等位基因的晚花植株，包括1个碱基对插入或短缺失。我们的结果表明，利用rgen划分组织靶向突变在拟南芥中提供了一种有效的可遗传基因组工程方法。本文的在线版本(doi：10.1007/s00425-0142180-5)包含补充材料，授权用户可以使用。
Dividing tissue-targeted site-directed mutagenesis using RGEN of CRISPR/Cas system produces heritable mutations in Arabidopsis thaliana. Site-directed genome engineering in higher plants has great potential for basic research and molecular breeding. Here, we describe a method for site-directed mutagenesis of the Arabidopsis nuclear genome that efficiently generates heritable mutations using the RNA-guided endonuclease (RGEN) derived from bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated) protein system. To induce mutagenesis in proliferating tissues during embryogenesis and throughout the plant life cycle, the single guide RNA (sgRNA) and Cas9 DNA endonuclease were expressed from the U6 snRNA and INCURVATA2 promoters, respectively. After Agrobacterium-mediated introduction of T-DNAs encoding RGENs that targets FLOWERING LOCUS T (FT) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 4 genes, somatic mutagenesis at the targeted loci was observed in T1 transformants. In the results of FT-RGEN, T1 plants often showed late flowering indicative of the presence of large somatic sectors in which the FT gene is mutated on both chromosomes. DNA sequencing analysis estimated that about 90 % of independent chromosomal DNA fragments carried mutations in the analyzed tissue of a T1 plant showing late flowering. The most frequently detected somatic polymorphism showed a high rate of inheritance in T2 plants, and inheritance of less frequent polymorphisms was also observed. As a result, late-flowering plants homozygous for novel, heritable null alleles of FT including a 1 bp insertion or short deletions were recovered in the following T2 and T3 generations. Our results demonstrate that dividing tissue-targeted mutagenesis using RGEN provides an efficient heritable genome engineering method in A. thaliana. The online version of this article (doi:10.1007/s00425-014-2180-5) contains supplementary material, which is available to authorized users.
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影响因子：
17.3
作者：
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通讯作者：
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