The deactivated CRISPR/Cas9 (dCas9) is now the most widely-used gene activators. However, the current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, CRISPR-assisted trans enhancer, for activating gene expression in high efficiency by combining dCas9-VP64/sgRNA with the widely used strong CMV enhancer. In this strategy, a CMV enhancer DNA was recruited to target gene in trans by two systems, dCas9-VP64/csgRNA-sCMV and dCas9-VP64-GLA4/sgRNA-UAS-CMV. The former recruited trans enhancer by the annealing between two short complementary oligonucleotides at the ends of sgRNA and trans enhancer. The latter recruited trans enhancer by the binding between GLA4 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 gene and variant endogenous genes in various cells, with much higher activation efficiency than the current dCas9 activators.
All data generated or analysed during this study are included in the manuscript and supporting files.
- Jinke Wang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Irwin Davidson, Institut de Génétique et de Biologie Moléculaire et Cellulaire, France
© 2019, Xu et al.
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