Fig. 5.

Engineered activatable promoters enable PPI-dependent conditional CRISPRa. (A) Schematic of different PPI-dependent CRISPRa systems. MCP-SoxS fusion is split and the two proteins are instead fused to one end of a heterodimerization domain. The heterodimerization domains used to build PPI-dependent CRISPRa systems are the SYNZIP5/SYNZIP6 pair, the ABA-responsive ABI/PYL1 domain, and the gibberellic acid (GA)-responsive GAI/GID1 domain. (B) Distance requirements of PPI-dependent CRISPRa. Left: Engineered promoter containing densely packed scRNA target sites and single base pair 5′ additions allows for CRISPRa targeting between −81 and −111 bp from the TSS. Right: Testing SYNZIP-CRISPRa between −81 and −91 bp from the TSS. SYNZIP-CRISPRa components are expressed at 5 nM. Fold change is calculated relative to an off-target scRNA for each promoter variant. (C) Tuning conditional CRISPRa response through titration of small molecule concentration. For ABA- and GA- CRISPRa, the corresponding small molecule was titrated between 0 and 10 or 0 and 10³ μM, respectively to find the optimal concentration. ABA- and GA-CRISPRa components are expressed at 10 nM. (D) Improving PPI-dependent and conditional CRISPRa response by optimizing component stoichiometries. The concentration of the plasmids expressing the MCP and SoxS components for each dimerization system was varied 1 to 25 nM and tested combinatorially to find the best ratio of the two heterodimers. ABA is added at 10 μM and GA is added at 10³ μM. Fold change is given relative to a reaction with no MCP and SoxS plasmids added.