Gene activation by a CRISPR-assisted trans enhancer.

Xinhui Xu,Danyang Wang,Jinliang Gao,Jinke Wang,Jian Wu,Wei Dai

doi:10.7554/elife.45973

Abstract

The deactivated CRISPR/Cas9 (dCas9) is now the most widely used gene activator. However, current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, the CRISPR-assisted trans enhancer, for activating gene expression at high efficiency by combining dCas9-VP64/sgRNA with the widely used strong CMV enhancer. In this strategy, CMV enhancer DNA was recruited to target genes in trans by two systems: dCas9-VP64/csgRNA-sCMV and dCas9-VP64-GAL4/sgRNA-UAS-CMV. The former recruited trans enhancer by annealing between two short complementary oligonucleotides at the ends of the sgRNA and trans enhancer. The latter recruited trans enhancer by binding between GAL4 fused to dCas9 and UAS sequence of trans enhancer. The trans enhancer activated gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter genes and variant endogenous genes in various cells, with much higher activation efficiency than that of current dCas9 activators.

Highlights

Clustered regularly interspaced short palindromic repeats (CRISPR) was originally identified in the immune system of bacteria, with the function of destroying the invading microphage DNA by enzymatic digestion
When dead Cas9 (dCas9) protein was guided to the promoter of the target gene by capture sgRNA (csgRNA), stick-end CMV (sCMV) could be recruited by csgRNA
Because the dCas9/csgRNA-anchored sCMV functions as a transcription factors in trans, we named it a trans enhancer to distinguish it from the natural cis enhancer

Summary

Introduction

Clustered regularly interspaced short palindromic repeats (CRISPR) was originally identified in the immune system of bacteria, with the function of destroying the invading microphage DNA by enzymatic digestion. The dead Cas (dCas9) and its associated single guide RNA (sgRNA) have been widely used to regulate gene expression in recent years (Dominguez et al, 2016; Hilton et al, 2015; Jinek et al, 2012; Kiani et al, 2015; Mali et al, 2013b; Radzisheuskaya et al, 2016; Wang et al, 2016) Both dCas and sgRNA have been engineered for activating or repressing gene expression. It is difficult to package most of these dCas fusion proteins into adeno-associated virus (AAV) for in vivo application

Methods

Results

Conclusion