A CRISPR/dCAS9 strategy to modify promoter activity: drought stress tolerance application

Abstract

Being sessile organisms, plants have to evolve towards a developmental and physiological flexibility in order to generate adaptation mechanisms against different biotic and abiotic stresses. In an agronomical aspect, these environmental factors impact negatively on plant development, representing an elevated economical cost. Water stress impacts different aspects of plant physiology, and can decrease their growth and productivity. Genetic manipulation of plant genomes can overcome some of these difficulties. The type II CRISPR/Cas system has been adapted in plants to control the genetic modification in a more targeted and precise procedure. The catalytically inactive Cas9 (dCas9) fused to activators/repressors has already been used to regulate transcription in transient transformed tobacco leaves and Arabidopsis plants. To improve the technique, we propose here to use the dCas9 fused to the tripartite activator VPR and two arabidopsis epigenetic modification domains: the Acetyltrasnferase domain from AtHAC1 (AT domain), and the methyltransferase domain from CURLY LEAF (CLF) gene (SET domain).  Our results showed that the dCas9 fused to VPR and AT increased the activity of promoters controlling a reporter gene. The dCas9 fused to the SET domain displayed contrasted results, sometimes activating and sometimes inhibiting the expression of the reporter gene. The strategy was also tested to control the endogene promoter of the transcription factor AtAREB1, known to control key genes in the response to drought stress. The AREB1 expression was increased in plants expressing the dCas9-VPR and dCas9-AT fusions, plants that showed a better tolerance to drought stress. Again, the results with the SET domain variated from one line to another, displaying activation and inhibition of the AREB1 expression, with opposed phenotypes when submitted to water withdrawal. Our data indicates that  targeted epigenetic modifications can be used to modify promoter activity. Here, we improved plant’s response to drought stress, but the approach can be designed to control any promoter.