Biology subjects

Bewg, W. P.

Publications and source records attributed to Bewg, W. P..

5 recordsLinked to original sources

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↗

bioRxiv2022

In vitro floral development in poplar: Insights into seed trichome and trimonoecy regulation

Woody perennials including Populus spp. (poplars) have a juvenile phase that ranges from several years to decades in length. This and the year-long floral development process are major impediments to breeding and to fundamental research of reproductive traits. Here we report a CRISPR-empowered in vitro flowering system and demonstrate its application using three reproductive traits: sex, seed trichomes, and a previously undescribed potential for trimonoecy in poplar.

plant biology↗

bioRxiv2022

Multiplex editing of the Nucleoredoxin1 tandem array in poplar: Decrypting outcomes due to CRISPR/Cas9-mediated translocations and complex inversions

The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. Here, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ~100 kb in Populus tremula x alba. We demonstrated efficient multiplex editing with one single gRNA in 42 transgenic lines. The mutation profiles ranged from small indels and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid functional characterization.

plant biology↗

bioRxiv2021

Multiplex knockout of trichome-regulating MYBs in poplar affects light sensitivity and triterpene accumulation

As the focus for CRISPR edited plants moves from proof-of-concept to real world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one gRNA per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in Populus tremula x P. alba INRA 717-1B4. Unexpected duplications of MYB186 and MYB138 resulted in a total of eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and PCR analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the non-glandular trichomes of poplar. ONE SENTENCE SUMMARYTargeting conserved sequences with a single gRNA allowed efficient mutagenesis of a multigene family and the recovery of trichomeless and triterpene-free poplar mutants.

plant biology↗