A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9

Lyujie Fang; Sandy S C Hung; Jennifer Yek; Layal El Wazan; Tu Nguyen; Shahnaz Khan; Shiang Y Lim; Alex W Hewitt; Raymond C B Wong

doi:10.1016/j.omtn.2018.11.008

A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9

Mol Ther Nucleic Acids. 2019 Mar 1:14:184-191. doi: 10.1016/j.omtn.2018.11.008. Epub 2018 Nov 20.

Authors

Lyujie Fang¹, Sandy S C Hung², Jennifer Yek³, Layal El Wazan³, Tu Nguyen³, Shahnaz Khan², Shiang Y Lim⁴, Alex W Hewitt⁵, Raymond C B Wong⁶

Affiliations

¹ Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia; Jinan University, Guangzhou, China.
² Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia.
³ Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia.
⁴ O'Brien Institute Department, St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.
⁵ Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Hobart, TAS, Australia.
⁶ Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia; Shenzhen Eye Hospital, School of Medicine, Shenzhen University, Shenzhen, China. Electronic address: wongcb@unimelb.edu.au.

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.

Keywords: CRISPR; CRISPR activation; SaCas9; SpCas9; cloning free; gene activation; multiplex gene activation; transcriptional activator.