title: Repurposing the CRISPR-Cas9 system for targeted DNA methylation.
abstract: Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.
authors.affiliation: Department of Biology, Division of Molecular Biology, University of Zagreb, Faculty of Science, Zagreb, HR-10000, Croatia.
authors.forename: Aleksandar
authors.identifier: 0000-0002-4627-1821
authors.initials: A
authors.lastname: Vojta
authors.affiliation: Department of Biology, Division of Molecular Biology, University of Zagreb, Faculty of Science, Zagreb, HR-10000, Croatia.
authors.forename: Paula
authors.initials: P
authors.lastname: Dobrinić
authors.affiliation: Department of Biology, Division of Molecular Biology, University of Zagreb, Faculty of Science, Zagreb, HR-10000, Croatia.
authors.forename: Vanja
authors.initials: V
authors.lastname: Tadić
authors.affiliation: Department of Biology, Division of Molecular Biology, University of Zagreb, Faculty of Science, Zagreb, HR-10000, Croatia.
authors.forename: Luka
authors.initials: L
authors.lastname: Bočkor
authors.affiliation: Department of Biology, Division of Molecular Biology, University of Zagreb, Faculty of Science, Zagreb, HR-10000, Croatia.
authors.forename: Petra
authors.initials: P
authors.lastname: Korać
authors.affiliation: Ragon Institute of MGHT, MIT and Harvard, 400 Technology Square, Cambridge, MA, USA.
authors.forename: Boris
authors.initials: B
authors.lastname: Julg
authors.affiliation: Department of Biology, Division of Molecular Biology, University of Zagreb, Faculty of Science, Zagreb, HR-10000, Croatia.
authors.forename: Marija
authors.initials: M
authors.lastname: Klasić
authors.affiliation: Department of Biology, Division of Molecular Biology, University of Zagreb, Faculty of Science, Zagreb, HR-10000, Croatia vzoldos@biol.pmf.hr.
authors.forename: Vlatka
authors.identifier: 0000-0002-1581-3720
authors.initials: V
authors.lastname: Zoldoš
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D050822:Cytokine Receptor gp130 / Q000235:genetics*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D000090205:DNA Methyltransferase 3A; D044127:Epigenesis, Genetic / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: C491123:BACH2 protein, human; D050976:Basic-Leucine Zipper Transcription Factors; C000717967:DNMT3A protein, human; C494199:IL6ST protein, human; D000094704:RNA, Guide, CRISPR-Cas Systems; D050822:Cytokine Receptor gp130; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A