Oncotarget

Research Papers:

Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system

Benjamin Garcia-Bloj, Colette Moses, Agustin Sgro, Janice Plani-Lam, Mahira Arooj, Ciara Duffy, Shreyas Thiruvengadam, Anabel Sorolla, Rabab Rashwan, Ricardo L. Mancera, Andrea Leisewitz, Theresa Swift-Scanlan, Alejandro H. Corvalan and Pilar Blancafort _

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Oncotarget. 2016; 7:60535-60554. https://doi.org/10.18632/oncotarget.11142

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Abstract

Benjamin Garcia-Bloj1,2,4, Colette Moses1,2, Agustin Sgro1, Janice Plani-Lam1, Mahira Arooj1,5, Ciara Duffy1,2, Shreyas Thiruvengadam1, Anabel Sorolla1, Rabab Rashwan1, Ricardo L. Mancera5, Andrea Leisewitz4, Theresa Swift-Scanlan6, Alejandro H. Corvalan3,4, Pilar Blancafort1,2

1Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia

2School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, WA, 6009, Australia

3Advanced Center for Chronic Diseases (ACCDiS), Pontificia Universidad Católica de Chile, Santiago, RM, 8330034, Chile

4Faculty of Medicine, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile

5School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth WA 6845, Australia

6School of Nursing, Virginia Commonwealth University, Richmond, Virginia, 23298, USA

Correspondence to:

Pilar Blancafort, email: pilar.blancafort@uwa.edu.au

Keywords: CRISPR/dCas9, ZF, TALE, tumor suppressor genes, gene reactivation

Received: June 01, 2016     Accepted: July 19, 2016     Published: August 09, 2016

ABSTRACT

The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.


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