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dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

Authors Duellman T, Doll A, Chen X, Wakamiya R, Yang J

Received 19 July 2017

Accepted for publication 30 August 2017

Published 19 September 2017 Volume 2017:4 Pages 63—73


Checked for plagiarism Yes

Review by Single-blind

Peer reviewer comments 3

Editor who approved publication: Dr Yoshifumi Itoh

Tyler Duellman, Andrea Doll, Xi Chen, Rie Wakamiya, Jay Yang

Department of Anesthesiology, University of Wisconsin SMPH, Madison, WI, USA

Abstract: Selective gene activation with the dCas9 (deactivated clustered regularly interspaced short palindromic repeats [CRISPR] associated protein 9)/CRISPR targeting of a transcriptional activator effector is now well established. However, the optimal targeting of guide RNA (gRNA) for a given gene is largely a matter of trial and error. We explored the optimal targeting site for tissue inhibitor of metalloproteinases (TIMPs) by first screening multiple gRNA target sites using a luciferase-based promoter-reporter system and next confirmed the effective TIMP induction in the mouse motor neuron-like neuron-enriched spinal cord 34 (NSC34) cells. Screening of many gRNAs targeting the 1–1.9 kB promoter regions of TIMP1–3 identified several hot-spots for optimal gene induction, however, no general pattern defining the optimal target site with respect to the proximity of known transcription factor binding sites or distance from the start ATG was apparent. TIMP2 with a larger basal transcriptional activity showed a greater fold-induction with gRNA compared with TIMP1 or 3 supporting the importance of an open-chromatin for best gRNA-mediated transcriptional induction. The rank order of induction potency for different gRNA identified in the promoter-reporter screening held true for the NSC34 cells. Co-activation with multiple gRNAs greatly increased the gene induction.

Keywords: TIMP, promoter, CRISPR, transcriptional activation, gRNA

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