Gene activation by a CRISPR-assisted enhancer.

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