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

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.

. Sequences of the A3 minimal promoters designed in this study for the GB_SynP collection. Highlighted in bold are the predicted TATA boxes of each promoter sequence. Underlined sequences corresponds to predicted 5' UTR regions.   GB3271 pUPD2_GB_SynP (A2) G1aG2b.7 A2 Proximal promoter sequence consisting of the target sequence for the gRNA-1 DFR (gRNA1) and gRNA-5 DFR (gRNA2) flanked by random sequences.

GB3319
Alpha1_R1:G1e.1:mDFR:luc:t35s TU for the expression of firefly luciferase driven by a GB_SynP promoter with A1 R1 and A3 mSlDFR parts, and an A2 part containing a target for the gRNA-1DFR (gRNA1, at "e site"). GB3320 Alpha1_R1:G3aG2b.1:mDFR:luc:t35s TU for the expression of firefly luciferase driven by a GB_SynP promoter with A1 R1 and A3 mSlDFR parts, and an A2 part containing a target for the gRNA-4 NOS (gRNA3) and gRNA-5 DFR (gRNA2). Module for the expression of luciferase driven by a GB_SynP promoter with A1 R1, A3 mSlDFR, and an A2 part with three times the target for gRNA1, and the constitutive expression of renilla and P19.

GB3408 pUPD2_mSlCHS
Minimal promoter of slCHS1 gene (Solyc09g091510) containing 62bp upstream the transcription start site and the 5'UTR region.

GB3409 pUPD2_mSlANS
Minimal promoter of slANS gene (Solyc10g076660) containing 62bp upstream the transcription start site and the 5'UTR region.

GB3410 pUPD2_mNtTA29
Minimal promoter of NtTA29 gene containing 62bp upstream the transcription start site and the 5'UTR region (from GB1477 sequence).

GB3411 pUPD2_mAt2S3
Minimal promoter of At2S3 gene containing 84bp upstream the transcription start site and the 5'UTR region (from GB0029 sequence).

GB3412 pUPD2_mNtPCPS2
Minimal promoter of NtPCPS2 gene containing 62bp upstream the transcription start site and the 5'UTR region (from GB1027 sequence).

GB3413 pUPD2_mPcPAF
Minimal promoter of PcPAF gene containing 62bp upstream the transcription start site and the 5'UTR region (from FB029 sequence).