GB_SynP: A Modular dCas9-Regulated Synthetic Promoter Collection for Fine-Tuned Recombinant Gene Expression in Plants

[Image: see text] Programmable transcriptional factors based on the CRISPR architecture are becoming commonly used in plants for endogenous gene regulation. In plants, a potent CRISPR tool for gene induction is the so-called dCasEV2.1 activation system, which has shown remarkable genome-wide specificity combined with a strong activation capacity. To explore the ability of dCasEV2.1 to act as a transactivator for orthogonal synthetic promoters, a collection of DNA parts was created (GB_SynP) for combinatorial synthetic promoter building. The collection includes (i) minimal promoter parts with the TATA box and 5′UTR regions, (ii) proximal parts containing single or multiple copies of the target sequence for the gRNA, thus functioning as regulatory cis boxes, and (iii) sequence-randomized distal parts that ensure the adequate length of the resulting promoter. A total of 35 promoters were assembled using the GB_SynP collection, showing in all cases minimal background and predictable activation levels depending on the proximal parts used. GB_SynP was also employed in a combinatorial expression analysis of an autoluminescence pathway in Nicotiana benthamiana, showing the value of this tool in extracting important biological information such as the determination of the limiting steps in an enzymatic pathway.