Deguchi, M. | bioRxiv Science

bioRxiv2024

A compendium of nonredundant short Polymerase III promoters for CRISPR applications

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 x 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.

plant biology↗

bioRxiv2024

CRISPR knockout of winter-biased SUT4 alters phenology, biomass accrual, and fertility of field-grown hybrid poplar

Climate uncertainty is intensifying the need for greater plasticity in carbohydrate reserve utilization to support winter survival and spring growth in woody perennials. In poplar, the single-copy SUT4, which encodes a tonoplast-localized sucrose transporter, and the SUT5/SUT6 genome duplicates, which encode plasma membrane-localized transporters, are expressed year-round, with SUT4 showing highest expression during cool seasons. Given its role in vacuolar sucrose efflux and winter-predominant expression, SUT4 may play a key role in modulating seasonal carbohydrate dynamics. While SUT4-knockdown and knockout effects have been studied under greenhouse conditions, their impact under field conditions remains unexplored. Here, we report a field-based study comparing CRISPR knockout mutants of winter-expressed SUT4 and SUT5/SUT6 in Populus tremula x alba. We show that sut4, but not sut56, mutants exhibited earlier autumn leaf senescence, delayed spring bud flush, reduced stem growth, and altered sugar partitioning in winter xylem and bark relative to controls. After two years in the field, all genotypes flowered before leaf flush in early spring; however, sut4 mutants produced sterile ovules despite developing normal-looking catkins. Metabolic profiling revealed disrupted sucrose and raffinose dynamics in elongating sut4 catkins, accompanied by transcriptomic signatures of elevated stress and downregulation of proanthocyanidin biosynthesis and circadian clock genes. These findings highlight the critical role of SUT4 in coordinating sugar allocation, stress responses, and seasonal development in poplar. Significance statementThis field study demonstrates that loss of SUT4, the most highly expressed sucrose transporter during cool seasons, disrupts phenology, growth, and fertility in poplar. Altered sugar and raffinose dynamics and transcriptomic signatures of stress and circadian clock gene dysregulation in the mutants underscore SUT4s role in coordinating sugar allocation and seasonal developmental transitions under natural environmental conditions.

plant biology↗