Laboratory for Genome Engineering, Division of Environmental and Biological Sciences and Engineering, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia.
Laboratory for Genome Engineering, Division of Environmental and Biological Sciences and Engineering, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia.
Virus Res. 2018 Jan 15;244:333-337. doi: 10.1016/j.virusres.2017.10.009. Epub 2017 Oct 16.
The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species. Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR 相关（Cas9）系统已实现了多种植物物种的高效基因组工程。然而，将基因组工程试剂（如单指导 RNA（sgRNA））递送至植物细胞仍然具有挑战性。在此，我们报告了烟草脆裂病毒（TRV）和豌豆早褐病毒（PEBV）的工程改造，以将一个或多个 sgRNA 递送至过表达含有 Cas9 的核定位信号的烟草原生质体和拟南芥（Col-0）植物。我们的数据表明，TRV 和 PEBV 可以将 sgRNA 递送至接种和系统叶片，并且这导致了靶向基因组位点的突变。此外，在烟草原生质体中，基于 PEBV 的 sgRNA 递送比基于 TRV 的递送导致更多的靶向突变。我们的数据表明，TRV 和 PEBV 可以促进植物基因组工程，并且可以用于产生靶向突变，用于功能分析和其他生物技术应用在不同的植物物种中。关键信息：将基因组工程试剂递送至植物细胞具有挑战性且效率低下，这限制了该技术在许多植物物种中的应用。RNA 病毒如 TRV 和 PEBV 为靶向基因组修饰的系统递送 sgRNA 提供了一种有效工具。
