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

// Benjamin Garcia-Bloj 1, 2, 4 , Colette Moses 1, 2 , Agustin Sgro 1 , Janice Plani-Lam 1 , Mahira Arooj 1, 5 , Ciara Duffy 1, 2 , Shreyas Thiruvengadam 1 , Anabel Sorolla 1 , Rabab Rashwan 1 , Ricardo L. Mancera 5 , Andrea Leisewitz 4 , Theresa Swift-Scanlan 6 , Alejandro H. Corvalan 3, 4 , Pilar Blancafort 1, 2 1 Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia 2 School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, WA, 6009, Australia 3 Advanced Center for Chronic Diseases (ACCDiS), Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile 4 Faculty of Medicine, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile 5 School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth WA 6845, Australia 6 School 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.