CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter

Abstract

// Samrat Roy Choudhury 1 , Yi Cui 1 , Katarzyna Lubecka 2 , Barbara Stefanska 2,3 , Joseph Irudayaraj 1,3 1 Department of Agricultural & Biological Engineering, Bindley Bioscience Centre, Purdue University, West Lafayette, IN 47907, USA 2 Department of Nutrition Science, Purdue University, West Lafayette, IN 47907, USA 3 Purdue Centre for Cancer Research, Purdue University, West Lafayette, IN 47907, USA Correspondence to: Barbara Stefanska, email: bstefanska@purdue.edu Joseph Irudayaraj, email: josephi@purdue.edu Keywords: CRISPR-dCas9, TET1, BRCA1, DNA demethylation, gene activation Received: March 10, 2016 Accepted: May 30, 2016 Published: June 23, 2016 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.