A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9
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2019-03-01
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Molecular Therapy: Nucleic Acids
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CELL PRESS
Melbourne Authors
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Surgery (St Vincent's)
Centre for Eye Research Australia (CERA)
Ophthalmology (Eye & Ear Hospital)
Centre for Eye Research Australia (CERA)
Ophthalmology (Eye & Ear Hospital)
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Journal Article
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Fang, L., Hung, S. S. C., Yek, J., El Wazan, L., Nguyen, T., Khan, S., Lim, S. Y., Hewitt, A. W. & Wong, R. C. B. (2019). A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9. Molecular Therapy Nucleic Acids, 14, pp.184-191. https://doi.org/10.1016/j.omtn.2018.11.008.
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Abstract
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.