Study : A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation
Identification
Name
A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation
Identifier
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Source
Description
CRISPR-based epigenome editing was recently used to activate gene expression through direct transcriptional activation or site-specific DNA demethylation. Viral delivery of guide RNAs for these purposes remains to be developed. Furthermore, currently available viral delivery tools for genome editing show meager rates of heritability. Here, we have developed a tobacco rattle virus (TRV)-based guide RNA delivery system for both transcriptional activation and targeted DNA demethylation. To promote heritable epigenome editing specifically within plant meristems and the germline, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome, thus facilitating cell-to-cell movement of the RNA in plants. We achieved up to ~8% heritability of the induced phenotype in the progeny of virus-inoculated plants and 25% in the following generation, indicating high rates of heritability for targeted DNA demethylation. Thus, TRV delivery, in combination with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing. Overall design: BS-seq and BS-PCR followed by sequencing were used to evaluate DNA methylation in plants expressing SunTag-TET1, with virally-supplied guide RNAs (+gRNAs) or no guide RNA sequence (-gRNAs; controls). Three different gRNAs (g4, g10, g18) were designed against several regions in the 5 region of the FWA locus and its promoter. Two Tobacco rattle virus vectors encoding a single guide RNA (g4) and multiple guide RNAs (g4, g10, and g18) were used to deliver the guide RNAs. BS-PCR was used to evaluate methylation at several regions of the FWA promoter in two pairs of +gRNA and matched -gRNA control lines, one at three days post-infection (3 dpi) and one at six dpi. BS-seq was later used to assess methylation in first- and second-generation descendants of these treated lines (2 +gRNAs and 2 -gRNA lines per generation), to evaluate changes both locally at FWA, and genome-wide.
Genotype
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