A compendium of nonredundant short Polymerase III promoters for CRISPR applications

Abstract Multiplex genome editing via CRISPR enables simultaneous gene knockout, activation, and repression, but current toolkits often rely on repetitive and lengthy Pol III promoters, limiting construct diversity and genetic stability. Here, we present a diverse collection of minimal U6 and U3 Pol III promoters that support efficient guide RNA (gRNA) expression in dicots. Using CRISPR-Cas9 editing of mEGFP in Nicotiana benthamiana and Populus tremula × alba, we demonstrate that minimal promoters can match the performance of their longer counterparts. A systematic evaluation of 14 short promoters revealed that most are functional across species, with exceptions linked to sequence variations in the upstream sequence element (USE) and TATA box. Mutagenesis confirmed the causal role of specific nucleotide changes in promoter activity. Furthermore, we show that regions outside these conserved motifs can be modified to create new-to-nature, functional promoters. This work refines the consensus for functional Pol III promoter elements and expands the toolkit for efficient, stable, and scalable CRISPR editing in dicots, with implications for synthetic promoter design. Competing Interest Statement The authors have declared no competing interest. Footnotes Revised figures, including additional data showing the application of short Pol III promoters in multiplex editing