Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta
Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta
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DOI：
10.1007/s11103-014-0266-x
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发表时间：
2015-01-01
影响因子：
5.1
通讯作者：
Levy, Avraham A.
中科院分区：
文献类型：
--
作者：
Johnson, Ross A.;Gurevich, Vyacheslav;Levy, Avraham A.
定制设计的核酸酶可以通过催化特定靶标处的DNA断裂以刺激靶向诱变或基因置换来实现精确的植物基因组编辑。CRISPR-Cas系统及其靶特异性RNA分子指导Cas9核酸酶，是对现有核酸酶的最新补充，这些核酸酶通过连接的蛋白质结构域（例如TALEN和锌指核酸酶）结合并切割靶。我们对这些不同类型的定制设计的核酸酶进行了比较研究，并评估了CRISPR-Cas系统的各种组件。为此目的，我们已经调整了我们先前报道的用于本氏烟草叶中的切割依赖性荧光素酶基因校正的测定（约翰逊等人，Plant Mol Biol 82（3）：207-221，2013）。我们发现通过CRISPR-Cas的切割比通过TALEN切割相同靶标更有效。我们还比较了基于使用三种不同Cas9基因变体的表达的化脓链球菌Cas9蛋白的切割效率。我们发现这些变体之间的切割效率存在显著差异，其中人类和拟南芥密码子优化的基因具有最高的切割效率。我们比较了12种全新设计的单一合成指导RNA（sgRNA）构建体的活性，发现当使用相同的Cas9核酸酶时，它们的切割效率变化很大。最后，我们表明，对于用我们的检测方法测试的靶标之一，我们可以在A. thaliana.这项工作强调了CRISPR-Cas系统在植物中的效率。它还表明，需要进一步的工作来预测sgRNA或Cas9变体的最佳设计。
Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.