Botanical Institute, Karlsruhe Institute of Technology, POB 6980, 76049, Karlsruhe, Germany.
Plant J. 2018 Jun;94(5):767-775. doi: 10.1111/tpj.13899. Epub 2018 May 4.
Application of the bacterial CRISPR/Cas systems to eukaryotes is revolutionizing biology. Cas9 and Cas12 (previously called Cpf1) are widely used as DNA nucleases for inducing site-specific DNA breaks for different kinds of genome engineering applications, and in their mutated forms as DNA-binding proteins to modify gene expression. Moreover, histone modifications, as well as cytosine methylation or base editing, were achieved with these systems in plants. Recently, with the discovery of the nuclease Cas13a (previously called C2c2), molecular biologists have obtained a system that enables sequence-specific cleavage of single-stranded RNA molecules. The latest experiments with this and also the alternative Cas13b system demonstrate that these proteins can be used in a similar manner in eukaryotes for RNA manipulation as Cas9 and Cas12 for DNA manipulations. The first application of Cas13a for post-transcriptional regulation of gene expression in plants has been reported. Recent results show that the system is also applicable for combating viral infection in plants. As single-stranded RNA viruses are by far the most abundant class of viruses in plants, the application of this system is of special promise for crops. More interesting applications are imminent for plant biologists, with nuclease dead versions of Cas13 enabling the ability to visualize RNA molecules in vivo, as well as to edit different kinds of RNA molecules at specific bases by deamination or to modify them by conjugation. Moreover, by combining DNA- and RNA-directed systems, the most complex of changes in plant metabolism might be achievable.
细菌 CRISPR/Cas 系统在真核生物中的应用正在彻底改变生物学。Cas9 和 Cas12(以前称为 Cpf1)被广泛用作 DNA 核酸酶,用于诱导各种基因组工程应用中的特定位置 DNA 断裂,并在其突变形式下作为 DNA 结合蛋白来修饰基因表达。此外,这些系统还可在植物中实现组蛋白修饰以及胞嘧啶甲基化或碱基编辑。最近,随着核酸酶 Cas13a(以前称为 C2c2)的发现,分子生物学家获得了一种能够特异性切割单链 RNA 分子的系统。最近的实验表明,该蛋白与 Cas13b 系统一样,可在真核生物中类似地用于 RNA 操作,就像 Cas9 和 Cas12 用于 DNA 操作一样。Cas13a 在植物中转录后基因表达调控中的首次应用已被报道。最近的结果表明,该系统也可用于植物抗病毒感染。由于单链 RNA 病毒是迄今为止植物中最丰富的病毒类群,因此该系统在作物中的应用具有特殊的前景。对于植物生物学家来说,更有趣的应用即将出现,Cas13 的无核酸酶版本使能够在体内可视化 RNA 分子,并通过脱氨作用在特定碱基处编辑不同类型的 RNA 分子,或通过缀合对其进行修饰。此外,通过结合 DNA 和 RNA 指导的系统,可能实现植物代谢中最复杂的变化。