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Site-specific manipulation of Arabidopsis loci using CRISPR-Cas9 SunTag systems


Understanding genomic functions requires site-specific manipulation of loci via efficient protein effector targeting systems. However, few approaches for targeted manipulation of the epigenome are available in plants. I have adapted and created a dCas9-SunTag system for use in plants. Here, we use the dCas9-SunTag system to target DNA demethylation. In addition, I have adapted the SunTag system to engineer targeted gene activation and targeted DNA methylation in Arabidopsis. We show that a SunTag system comprising of the TET1 catalytic domain is able to demethylate the promoter of FWA and regions of the CACTA1 transposon, leading to changes in gene expression. I demonstrate that a dCas9-SunTag system utilizing the transcriptional activator VP64 drives robust and specific activation of several loci, including protein coding genes and transposable elements, in diverse chromatin contexts. In addition, I present a SunTag system with the catalytic domain of the Nicotiana tabacum DRM methyltransferase, which efficiently targets DNA methylation to specific loci, including the FWA promoter, triggering a developmental phenotype, and the SUPERMAN promoter. These SunTag systems represent valuable tools for the site-specific manipulation of plant epigenomes.

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