Synthetic Biology (SynBio) aims at rewiring plant metabolic and developmental programs with orthogonal regulatory circuits. This endeavour requires new molecular tools able to interact with endogenous factors in a potent yet at the same time highly specific manner. A promising new class of SynBio tools that could play this function are the synthetic transcriptional activators based on CRISPR/Cas9 architecture, which combine autonomous transcriptional activation domains (TADs) capable of recruiting the cellular transcription machinery, with the easily customizable DNA‐binding activity of nuclease‐inactivated Cas9 protein (dCas9), creating so‐called Programmable Transcriptional Activators (PTAs). Initial dCas9‐PTAs versions comprising single domain fusions to Cas9 showed only low/moderate activation rates in plants. A new generation PTAs developed for mammalian cells combine several TADs attached to a single dCas9 protein, reaching higher activation rates. Multi‐TAD display can be achieved following different strategies (see Figure 1b). The SunTag strategy (Tanenbaum et al. 2014) uses multi‐epitope tags to attach multiple TADs to the dCas9 protein, whereas SAM and scRNA strategies (Mali et al. 2013, Konermann et al. 2015) use RNA aptamers added to the gRNA scaffold as secondary anchoring sites for TADs. Recently, some multi‐TAD designs have been successfully applied to plants (Li et al. 2017, Lowder et al. 2018), however a comprehensive multi‐parallel test for plant dCas9‐PTAs including multi‐TAD designs is still missing, leaving the door open for design improvements. Here we show a systematic comparison of 43 SunTag, SAM and scRNA‐based TAD combinations tested for their ability to activate different promoters fused to a Luciferase reporter. 

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