A Streamlined CRISPRa Architecture with Dual Pol II/Pol III Promoter and Optimized scRNA Enables Robust and Tunable...

CRISPR activation (CRISPRa) offers a powerful approach to upregulate endogenous genes; yet, existing systems in plants can be complex or difficult to integrate with CRISPR interference (CRISPRi). Here, we present a streamlined and flexible CRISPRa platform that enables robust gene activation. Using a dual-luciferase reporter, we benchmarked a range of guide RNA scaffolds, effector proteins, and promoters. We developed a novel single-guide RNA (sgRNA) architecture, harboring two MS2 aptamers inserted into the tetraloop and driven by a composite Pol II/Pol III promoter, as the most efficient configuration. This scaffold outperformed gR2.0- and SunTag-based constructs, reaching up to 100-fold activation of a minimal 35S promoter and up to 215-fold induction of three endogenous rice genes in protoplast assays. In contrast, scaffold RNAs (scRNAs) with aptamers at the 3' end or in excessive copy numbers were ineffective. Exploratory AlphaFold modeling supports a possible role for aptamer positioning and MCP-VP64 dimerization, although this remains a working hypothesis. This modular design enables tunable gene regulation in rice protoplasts and provides a practical platform for high-throughput screening and synthetic gene circuit prototyping in plants. Given that scRNA geometry and promoter architecture are universal features of CRISPR-based transcriptional modulation, the system is expected to be broadly portable across species. While the architecture is intended to be compatible with CRISPRi, future studies will be needed to establish its practical use in combined CRISPRa/i settings.