High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns.
High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns.
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由包含多个内含子的Cas9基因介导的植物中的高效基因组编辑。
DOI：
10.1016/j.xplc.2020.100135
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发表时间：
2021-03-08
影响因子：
10.5
通讯作者：
Marillonnet S
中科院分区：
文献类型：
--
作者：
Grützner R;Martin P;Horn C;Mortensen S;Cram EJ;Lee-Parsons CWT;Stuttmann J;Marillonnet S
最近发现的CRISPR/Cas9系统的作用模式为生物学家提供了一种有用的工具，可以在感兴趣的基因中产生位点特异性突变。在植物中，位点靶向突变通常是通过将Cas9表达构建体稳定地转化到植物基因组中获得的。在目标基因中引入突变的效率取决于构建体的具体特征，包括用于表达Cas9基因和引导RNA的启动子和终止子的来源和性质，以及Cas9核酸酶本身的序列。为了优化拟南芥Cas9核酸酶产生靶基因突变的效率，我们研究了其核苷酸和/或氨基酸序列的几个特征，包括密码子的使用、核定位信号（NLSs）的数量以及内含子的存在与否。我们发现Cas9基因密码子的使用对其活性有一定的影响，两个NLSs比一个NLSs效果更好。然而，通过在Cas9编码序列中添加13个内含子，这些构建体的编辑效率达到了最高，这大大提高了构建体的编辑效率。缺乏内含子的Cas9基因获得的原代转化子均未显示出敲除突变表型，而内含子Cas9基因产生的原代转化子中有70%至100%显示出突变表型。内含子化的Cas9基因也被发现在其他植物中有效，如烟叶和玫瑰花。CRISPR/Cas9系统已被广泛用于产生感兴趣的靶基因突变，但当使用不同的Cas9基因编码序列时，其效率不同。本研究表明，在拟南芥Cas9编码序列中引入多个内含子可以显著提高Cas9基因的效率。内含的Cas9基因在其他植物物种中也有很好的作用。
The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana, we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus. The CRISPR/Cas9 system has been widely used for generating mutations in target genes of interest, but with different efficiencies when different Cas9 gene coding sequences are used. This study shows that the introduction of multiple introns into the Cas9 coding sequence can dramatically increase the efficiency of the Cas9 gene in Arabidopsis thaliana. The intronized Cas9 gene also works well in other plant species.
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