A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana

• Published in: PLoS genetics Vol. 16; no. 12; p. e1008983
• Main Authors: Ghoshal, Basudev, Vong, Brandon, Picard, Colette L., Feng, Suhua, Tam, Janet M., Jacobsen, Steven E.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14.12.2020; Public Library of Science (PLoS)
• Subjects: Arabidopsis; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Biology and life sciences; CRISPR; CRISPR-Cas Systems; Demethylation; Deoxyribonucleic acid; Diseases and pests; Distribution; DNA; DNA Methylation; Drug delivery systems; Drugs; Engineering and Technology; Epigenetics; Epigenome; Experiments; Gene Editing - methods; Gene expression; Gene regulation; Gene Targeting - methods; Genetic aspects; Genomes; Innovations; Plant Viruses - genetics; Proteins; Repressors; Research and Analysis Methods; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Transfer - genetics; Tobacco mosaic virus; Transcription activation; Transcription factors; Transcriptional Activation; Transcriptional coactivators; Transgenic plants; Vectors (Biology); Vehicles; Viruses; United States
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1008983

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.