title: CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter.
abstract: DNA hypermethylation at the promoter of tumour-suppressor genes is tightly correlated with their transcriptional repression and recognized as the hallmark of majority of cancers. Epigenetic silencing of tumour suppressor genes impairs their cellular functions and activates a cascade of events driving cell transformation and cancer progression. Here, we examine site-specific and spatiotemporal alteration in DNA methylation at a target region in BRCA1 gene promoter, a model tumour suppressor gene. We have developed a programmable CRISPR-Cas9 based demethylase tool containing the deactivated Cas9 (dCas9) fused to the catalytic domain (CD) of Ten-Eleven Translocation (TET) dioxygenase1 (TET1CD). The fusion protein selectively demethylates targeted regions within BRCA1 promoter as directed by the designed single-guide RNAs (sgRNA), leading to the transcriptional up-regulation of the gene. We also noticed the increment in 5-hydroxymethylation content (5-hmC) at the target DNA site undergoing the most profound demethylation. It confirms the catalytic activity of TET1 in TET1-dCas9 fusion proteins-mediated demethylation at these target sequences. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin or DNA modifying enzymes and for loci-specific targeting to uncover epigenetic regulatory pathways at gene promoters and other selected genomic regions.
authors.affiliation: Department of Agricultural & Biological Engineering, Bindley Bioscience Centre, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Samrat Roy
authors.initials: SR
authors.lastname: Choudhury
authors.affiliation: Department of Agricultural & Biological Engineering, Bindley Bioscience Centre, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Department of Nutrition Science, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Lubecka
authors.affiliation: Department of Nutrition Science, Purdue University, West Lafayette, IN 47907, USA.|Purdue Centre for Cancer Research, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Stefanska
authors.affiliation: Department of Agricultural & Biological Engineering, Bindley Bioscience Centre, Purdue University, West Lafayette, IN 47907, USA.|Purdue Centre for Cancer Research, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Irudayaraj
mesh_terms: D064113:CRISPR-Cas Systems / Q000502:physiology*; D020134:Catalytic Domain; D018899:CpG Islands; D019175:DNA Methylation; D019398:Genes, BRCA1; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D006899:Mixed Function Oxygenases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics*
chemical_list: D011518:Proto-Oncogene Proteins; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human
keywords: BRCA1; CRISPR-dCas9; DNA demethylation; TET1; gene activation