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A simple cloning-free method to efficiently induce gene expression using CRISPR/Cas9


Fang, L and Hung, SSC and Yek, J and El Wazan, L and Nguyen, T and Khan, S and Lim, SY and Hewitt, AW and Wong, RCB, A simple cloning-free method to efficiently induce gene expression using CRISPR/Cas9, Molecular Therapy - Nucleic Acids, 14 pp. 184-191. ISSN 2162-2531 (2019) [Refereed Article]


Copyright Statement

Copyright 2018 The Authors. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

DOI: doi:10.1016/j.omtn.2018.11.008


Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular- and cell-biology disciplines.

Item Details

Item Type:Refereed Article
Keywords:CRISPR, CRISPR activation, SaCas9, SpCas9, cloning free, gene activation, multiplex gene activation, transcriptional activator
Research Division:Biomedical and Clinical Sciences
Research Group:Ophthalmology and optometry
Research Field:Vision science
Objective Division:Health
Objective Group:Clinical health
Objective Field:Clinical health not elsewhere classified
UTAS Author:Hewitt, AW (Professor Alex Hewitt)
ID Code:132283
Year Published:2019
Web of Science® Times Cited:9
Deposited By:Menzies Institute for Medical Research
Deposited On:2019-05-01
Last Modified:2020-03-11
Downloads:19 View Download Statistics

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