Simulations Predict Stronger CRISPRi Transcriptional Repression in Plants for Identical than Heterogeneous gRNA Target Sites

Plant synthetic biologists have been working to adapt the CRISPRa and CRISPRi promoter regulation methods for applications such as improving crops or installing other valuable pathways. With other organisms, strong transcriptional control has typically required multiple gRNA target sites, which poses a critical engineering choice between heterogeneous sites, which allow each gRNA to target existing locations in a promoter, and identical sites, which typically require modification of the promoter. Here, we investigate the consequences of this choice for CRISPRi plant promoter regulation via simulation-based analysis, using model parameters based on single gRNA regulation and constitutive promoters in N. benthamiana. Using models of 2 to 6 gRNA target sites to compare heterogeneous versus identical sites for tunability, sensitivity to parameter values, and sensitivity to cell-to-cell variation, we find that identical gRNA target sites are predicted to yield far more effective transcriptional repression than heterogeneous sites.