Gene activation by a CRISPR-assisted trans enhancer

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 <i>trans</i> 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 <i>trans</i> by two systems: dCas9-VP64/csgRNA-sCMV and dCas9-VP64-GAL4/sgRNA-UAS-CMV. The former recruited <i>trans</i> enhancer by annealing between two short complementary oligonucleotides at the ends of the sgRNA and <i>trans</i> enhancer. The latter recruited <i>trans</i> enhancer by binding between GAL4 fused to dCas9 and UAS sequence of <i>trans</i> enhancer. The <i>trans</i> enhancer activated gene transcription as the natural looped <i>cis</i> enhancer. The <i>trans</i> 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.