Oncotarget

Research Papers:

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

Samrat Roy Choudhury, Yi Cui, Katarzyna Lubecka, Barbara Stefanska and Joseph Irudayaraj _

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Oncotarget. 2016; 7:46545-46556. https://doi.org/10.18632/oncotarget.10234

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Abstract

Samrat Roy Choudhury1, Yi Cui1, Katarzyna Lubecka2, Barbara Stefanska2,3, Joseph Irudayaraj1,3

1Department of Agricultural & Biological Engineering, Bindley Bioscience Centre, Purdue University, West Lafayette, IN 47907, USA

2Department of Nutrition Science, Purdue University, West Lafayette, IN 47907, USA

3Purdue 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.


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