A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

Abstract

CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations in place, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold for the endogenous N. benthamiana DFR gene, with negligible expression in the absence of the trigger. The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

GB_SynP: a modular dCas9-regulated synthetic promoter collection for fine-tuned recombinant gene expression in plants

“…Owing to its proven responsiveness to dCasEV2.1, and especially the low basal expression levels observed in repeated experiments, we decided to use the p SlDFR structure as the basis for the design of a new set of dCasEV2.1-regulated synthetic promoters (Figure 1C). A “minimal promoter” element was designed by selecting the region comprising the 5’UTR and the TATA box from the SlDFR gene (named mDFR) as previously reported by Garcia-Perez et al 21 . This element was assigned a standard A3(−B2) position, according to the Phytobrick syntax, thus being flanked by TCCC and AATG overhangs (Figure 1A).…”

“…These results position the GB_SynP collection as a promising tool for the regulation of complex multigene circuits with different gRNAs present in the cis-regulatory boxes of each promoter, thus creating logic gates that could be useful to further explore different metabolic fluxes within biosynthetic pathways. Moreover, other studies reported the successful expression of gRNAs under pol-II promoters, which in turn could be regulated by different inducers 21,27 , thus allowing customizable control of each gRNA with different stimuli to further direct the multigene circuits in different ways.…”

“…While we reported here the assembly and behaviour of 35 synthetic promoters, the GB_SynP collection includes to date 32 promoter parts, compiled in Table S2, that can be used to assemble more than 500 different promoters without the need for creating any new sequence, standing out as one of the CRISPRa-based synthetic expression tools currently available for plants with the highest diversity and combinatorial strength. Moreover, plant synthetic promoters created so far mostly rely on the well-characterised CaMV 35s minimal promoter 27,30 , which might lead to higher basal expression in comparison with other minimal promoters like mDFR 21 . To overcome this limitation, GB_SynP includes newly designed minimal promoter parts, for which negligible basal expression was shown in all cases, as well as a range of activation levels.…”