PLOS Genetics|植物表观基因组编辑系统来了！
植物RNA病毒因其在病毒增殖和移动过程中大量积累和高效传播而被用作传递载体。利用这一概念，已经开发了几种用于CRISPR-Cas9基因组编辑的基于病毒的引导RNA传递平台。CRISPR-Cas9系统也适用于表观基因组编辑。虽然针对CRISPR-Cas9基因激活或位点特异性DNA去甲基化的系统已经开发出来，但针对这些目的的病毒导盲rna传递仍有待开发。
图 1. TRV接种植物的后代中DNA脱甲基和FWA表达进一步增强
为了填补这一空白，我们开发了一种基于TRV的烟草拨铃病毒(tobacco rattle virus, TRV)的单导RNA传递系统，用于拟南芥的表观基因组编辑。
由于tRNA-like序列已被证明可促进植物中RNA在细胞间的移动，我们使用tRNA-guide RNA表达系统从病毒基因组中表达guide RNAs，以促进可遗传的表观基因组编辑。我们证明了tRNA-gRNA系统可以用于拟南芥花期FLOWERING WAGENINGEN基因的转录激活和靶向DNA去甲基化。我们在接种病毒的植株后代中获得了高达8%的诱导去甲基化表型的遗传率。我们在下一代中没有检测到病毒，这表明病毒从植物组织中被有效清除。
因此，TRV的传递，结合特定的tRNA-gRNA架构，提供了快速和有效的表观基因组编辑。
图 2. TRV介导的SunTag-VP64植物中的RNA引导引起FWA的转录激活。
Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.
Ghoshal B, Vong B, Picard CL, Feng S, Tam JM, Jacobsen SE (2020) A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana. PLoS Genet 16(12): e1008983. https://doi.org/10.1371/journal.pgen.1008983
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