Abstract
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.
Highlights
Tools that enable targeted regulation of gene expression provide powerful systems for studying and manipulating diverse cellular processes [1]
We performed an initial set of experiments to test whether this tobacco rattle virus (TRV) based guide RNA delivery system could be used for transcriptional activation
We found that the targeted DNA demethylation can be heritable, even though viral RNAs are no longer detectable in progeny plants
Summary
Tools that enable targeted regulation of gene expression provide powerful systems for studying and manipulating diverse cellular processes [1]. Targeted gene regulation at the transcriptional level can be achieved by recruiting transcriptional activators, repressors, or epigenetic modifiers to a particular genomic locus using a programmable DNA-binding module, such as the catalytically inactive version of the CRISPR-CAS9 system [1,2,3]. Inactive CAS9 (dCas9) can act as a DNA binding module, enabling the targeted recruitment of any effector protein attached to dCas. Inactive CAS9 (dCas9) can act as a DNA binding module, enabling the targeted recruitment of any effector protein attached to dCas9 Such CRISPR-dCAS9 based tools have been used to regulate gene expression and/or modify the epigenome across multiple species [3,6,7,8,9]
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