Biology subjects

Lemaux, P. G.

Publications and source records attributed to Lemaux, P. G..

6 recordsLinked to original sources

bioRxiv2026

Targeted knockout of CYP79A1 reduces cyanogenic potential in grain sorghum

Sorghum bicolor is a climate-resilient C4 crop used for food, forage, and bioenergy, but broader adoption is constrained by accumulation of the cyanogenic glucoside dhurrin, which can release toxic hydrogen cyanide (HCN) upon tissue damage. Dhurrin levels are especially high in juvenile tissues, creating risk for grazing animals and limiting use in mixed crop-livestock systems. Here, we establish a CRISPR-Cas9 genome-editing strategy targeting CYP79A1, which catalyzes the first committed step in dhurrin biosynthesis, in the elite grain sorghum inbred RTx430, yielding transgene-free lines with stable, heritable reduction in cyanogenic potential across early vegetative development. Homozygous cyp79a1 knockouts were negligibly cyanogenic, whereas heterozygous plants exhibited approximately half the cyanogenic potential of unedited nulls. Consistent with established livestock grazing guidelines, only homozygous knockouts fell below thresholds considered hazardous for incidental grazing. This work establishes CYP79A1 as a practical and heritable genome-editing target for reducing sorghum cyanogenesis and provides a clear path for deployment of low-cyanogenic alleles in elite breeding backgrounds.

plant biology↗

bioRxiv2026

Architecting cis-regulation to quantitatively tune gene expression in cereals

Precise modulation of gene expression via cis-regulatory editing holds promise for non-transgenic crop improvement, but the sequence-to-function relationships that govern plant promoter activity remain poorly understood. Here, we develop a massively parallel reporter assay (MPRA) in Sorghum bicolor to systematically measure the effects of >30,000 CRISPR-like mutations-deletions, substitutions, and motif insertions-across entire native promoters and 5' untranslated regions (UTRs) of three photosynthesis genes: PsbS, Raf1, and SBPase. We find that gene expression is most tunable within a [~]500 base pair core promoter region, where mutational effects are reproducible across biological replicates and predictive of protein output. Within these regions, we identify compact deletions and motif insertions that strongly increase protein production (>30-fold relative to wild type), exceeding the performance of transgenic enhancer elements. Mutation-effect relationships are gene-specific, highlighting the need for tailored regulatory maps. Our results establish a high-throughput strategy for cis-regulatory fine-mapping that enables crop improvements via minimal, precise, and non-transgenic gene edits.

synthetic biology↗

bioRxiv2025

Multi-season analysis reveals hundreds of drought-responsive genes in sorghum

Persistent drought affects global crop production and is becoming more severe in many parts of the world in recent decades. Deciphering how plants respond to drought will facilitate the development of flexible mitigation strategies. Sorghum bicolor L. Moench (sorghum), a major cereal crop and an emerging bioenergy crop, exhibits remarkable resilience to drought. To better understand the molecular traits that underlie sorghums remarkable drought tolerance, we undertook a large-scale sorghum gene expression profiling effort, totaling nearly 1,500 transcriptome profiles, across a 3-year field study with replicated plots in Californias Central Valley. This study included time-resolved gene expression data from roots and leaves of two sorghum genotypes, BTx642 and RTx430, with different pre-flowering and post-flowering drought-tolerance adaptations under control and drought conditions. Quantification of genotype-specific drought tolerance effects was enabled by de novo sequencing, assembly, and annotation of both BTx642 and RTx430 genomes. These reference-quality genomes were used to construct a pan-gene set for characterizing conserved and genotype-specific expression. By integrating time-resolved transcriptomic responses to drought in the field across three consecutive years, we identified a set of drought-responsive genes that responded similarly in all three years of our field study. This expansive dataset represents a unique resource for sorghum and drought research communities and provides a methodological framework for the integration of multi-faceted time-resolved transcriptomic datasets.

genomics↗

bioRxiv2025

High-frequency Wus2-integrated transformation toolkit enhances Cas9 editing efficiency and expands capability with SpRY in sorghum

Sorghum bicolor L. (Moench), the fifth most important cereal crop internationally, is used as food, feed, forage, and fuels. Importantly, sorghums natural tolerance to environmental stresses leads to tolerance to climate variation. To optimize sorghum germplasm for crop improvement requires highly efficient genetic transformation and genome editing; however, sorghum has historically been recalcitrant to these genetic approaches. In this study, we report a high-efficiency engineering toolkit, optimized for genome editing via Agrobacterium-mediated transformation. Using CRISPR/Cas9-based editing machinery, the genetic tools led to editing efficiencies up to 95.7% when monocistronic guide RNAs targeted the phytoene desaturase (SbPDS) gene. We also tested a PAM (protospacer adjacent motif)-broadened Cas9 variant, intronized SpRY (ZmSpRYi), achievling comparable editing efficiencies. Using this toolkit permits exploitation of advanced editing tools, like prime editors, base editors, and targeted knock-in methods. These genetic advances offer new methods for sustainable crop improvement in sorghum and potentially other cereals. Technology readinessThis study demonstrates the development of an integrated genome engineering platform that combines morphogenic gene-assisted transformation with CRISPR/Cas9- and SpRY-mediated mutagenesis for multiplex genome editing, establishing a Technology Readiness Level (TRL) of 4/5. This workflow enables the generation of allelic variants through editing of both functional and regulatory elements in the plant genome. Advancing toward TRL 5 will require additional validation in relevant sorghum genotypes, including elite varieties. Moreover, the PAM-flexible editor SpRY is still under optimization, particularly at non-canonical PAM sites, which may limit its application for the modification of certain cis-regulatory elements. Further improvements in precision genome editing, such as the integration of prime editing or targeted knock-in technologies, will also be necessary to enable desired and predictable modifications. With continued optimization and incorporation of these advanced tools, this robust genome engineering platform has the potential to accelerate plant breeding and enable basic research supporting plant-based solutions to address climate change.

plant biology↗

bioRxiv2021

The recent evolutionary rescue of a staple crop depended on over half a century of global germplasm exchange

Rapid environmental change can lead to extinction of populations or evolutionary rescue via genetic adaptation. In the past several years, smallholder and commercial cultivation of sorghum (Sorghum bicolor), a global cereal and forage crop, has been threatened by a global outbreak of an aggressive new biotype of sugarcane aphid (SCA; Melanaphis sacchari). Here we characterized genomic signatures of adaptation in a Haitian sorghum breeding population, which had been recently founded from admixed global germplasm, extensively intercrossed, and subjected to intense selection under SCA infestation. We conducted evolutionary population genomics analyses of 296 post-selection Haitian lines compared to 767 global accessions at 159,683 single nucleotide polymorphisms. Despite intense selection, the Haitian population retains high nucleotide diversity through much of the genome due to diverse founders and an intercrossing strategy. A genome-wide fixation (FST) scan and geographic analyses suggests that adaptation to SCA in Haiti is conferred by a globally-rare East African allele of RMES1, which has also spread to other breeding programs in Africa, Asia, and the Americas. De novo genome sequencing data for SCA resistant and susceptible lines revealed putative causative variants at RMES1. Convenient low-cost markers were developed from the RMES1 selective sweep and successfully predicted resistance in independent U.S. x African breeding lines and eight U.S. commercial and public breeding programs, demonstrating the global relevance of the findings. Together, the findings highlight the potential of evolutionary genomics to develop adaptive trait breeding technology and the value of global germplasm exchange to facilitate evolutionary rescue.

evolutionary biology↗

bioRxiv2020

Pathway to Validate Gene Function in Key Bioenergy Crop, Sorghum bicolor

Determining gene function is an essential goal for the key bioenergy crop, Sorghum bicolor (L.) Moench - particularly for genes associated with its notable abiotic stress tolerances. However, detailed molecular understanding of the genes associated with those traits is limited. This was made clear in our in-depth transcriptome studies in sorghum, which indicated nearly 50% of its transcriptome is not annotated. In this report, we describe a full spectrum of tools needed to transform sorghum in order to validate and annotate genes. Efforts began with modifying a transformation method that uses the morphogenic genes Baby Boom and Wuschel2 (Ovule Development Protein2) to accelerate transformation speed and expand amenable genotypes. In our experience, transforming RTx430 without morphogenic genes requires ~18 to 21 weeks, compared with ~10 to 12 weeks to generate T0 plants using methods with morphogenic genes. Utilizing morphogenic genes also allowed for the transformation of several sorghum genotypes not previously transformed or historically recalcitrant to transformation, i.e., rapid cycling SC187, stay-green BTx642, BTx623 and sweet sorghum Ramada. In order to validate candidate genes via engineering, while simultaneously introducing the morphogenic genes, a co-transformation strategy, termed altruistic transformation, was developed. To accomplish editing of the target gene, phytoene desaturase, novel constructs were created that also included morphogenic genes. To enable full characterization of transformed plants, we adapted techniques to determine copy number and independence of events at high-throughput levels. Through these efforts, we created a complete pathway from Agrobacterium infection to high-throughput molecular genotyping that can be used to ascertain gene function and expedite basic genetic research in this widely-grown bioenergy crop plant.

plant biology↗