Development of CRISPRi Orthogonal Repression Systems in Plant Cells Using Synthetic Variants of the Figwort Mosaic Virus 34S Promoter with Two Identical sgRNA Binding Sites

The plant synthetic biology toolbox is rapidly expanding; however, there are still limited options for engineering logic gates for the precise modulation of gene expression. CRISPR interference (CRISPRi) represents a promising strategy for engineering logic into plant cells; however, only a limited number of promoter modules have been characterized for CRISPRi-mediated repression. In this study, the transient transgene expression in agroinfiltrated Nicotiana benthamiana leaves was used to assess the repressibility of a number of promoters with different strengths, including the Figwort Mosaic Virus (FMV) 34S promoter, which showed high repression efficiency using CRISPRi. Using dCas9 fused to the SRDX repressor domain, we employed single and double (identical or heterogeneous) sgRNA strategies for evaluating the repressibility of a library of 33 variants of the 34S promoter. This investigation supported a previous computer simulation predicting that a promoter with identical sgRNA binding sites is more efficiently repressed than a counterpart with heterogeneous sites; however, the repression efficiency varied, depending on the binding site location within the target promoter. In a second step, the top-performing 34S mutant/sgRNA/dCas9-repressor was used in combination with a Cre/loxP RNA scaffold orthogonal system to design a genetic switch, providing a versatile tool for modulation of gene expression. These results provide valuable perspectives on the utilization of 34S promoter modules in plant synthetic biology and the design of valuable CRISPRi genetic tools for precise modulation of transgene expression.