dSaCas9 enables enhanced transcriptional activation in Nicotiana benthamiana compared to its dSpCas9 ortholog

The SaCas9 from Staphylococcus aureus has been shown to be more effective than the commonly used SpCas9 from Streptococcus pyogenes for the generation of mutations in plants and has boosted CRISPR/Cas systems. CRISPRa/Cas systems on the other hand have been focused on the use of SpCas9. Building on foundational work, we developed a modular CRISPRa/Cas ortholog system based on a single plasmid infiltration for the use and comparison of both SaCas9 and SpCas9 for enhanced gene expression. Utilizing the multi-kingdom Golden Gate cloning platform, we integrated various elements to create a widely adaptable system. For the first time in the current study, we demonstrate that the dSaCas9 effector induces stronger activation of reporter gene expression by targeted promoters than dSpCas9 in plants. As both effectors are sharing a common target site for the tested promoter, they could be directly compared with each other showing that the dSaCas9-effector exhibited a stronger effect in the expression of the reporter genes. This allows fine-tuning the expression of genes by using different effectors. In addition, we could show on the example of the pNOS promoter and a truncated version of the pNOS promoter, that a lower basic expression can lead to an increased relative induction.