Biology subjects

Voytas, D.

Publications and source records attributed to Voytas, D..

9 recordsLinked to original sources

bioRxiv2026

Engineering carotenoid and steroidal glycoalkaloid depleted tomato fruit for heterologous production of high value terpenes

Plants produce specialized metabolites that function in plant defense and as attractants to pollinators and symbionts. One class of specialized metabolites are terpenoids that are synthesized from universal C5 building blocks via activities including terpene synthases, cytochromes P450, and glycosyl transferases. Some terpenes are highly valued for their use as insect repellants, fragrances, antimicrobial compounds, low calorie sweeteners, flavors, and medicines. Low abundance in target tissues, present in complex mixtures, as well as challenging extraction logistics are barriers to economic sustainable production of these compounds from their native species. While heterologous expression of terpenoid biosynthetic genes is feasible, the potential derivation of the products into conjugates via endogenous cytochromes P450 and glycosyl transferases limits this approach. In this project, we used multiplex gene editing technologies to overcome these challenges by creating novel tomato chassis with altered terpenoid biosynthetic capacity in fruit. Excluding central metabolic genes to minimalize impacts on growth and development, we selected 23 known and potential terpene-related genes expressed specifically in the fruit for gene editing. Fruit production and metabolic profiles of three chassis lines with alterations in the major classes of fruit specialized metabolites indicate loss of these genes is tolerated for fruit production. These combinatorial knockouts also showed modulation of native carbon reallocation toward endogenous sinks beneficial for a biosynthetic chassis. Establishing metabolite-modified fruit chassis demonstrates efficient combinatorial editing of entire branches of plant specialized metabolism, facilitating engineering of heterologous terpenes of industrial interest in tomato fruit.

plant biology↗

bioRxiv2026

A Tissue Culture Free Genome Editing Strategy in Plants Using Broad-Host-Range Viral Vectors Derived from Geminiviruses

The use of viral vectors offers a promising alternative to traditional transformation methods for creating gene-edited plants. In this study, we developed a novel plant genome editing system by delivering Cas9, Cas12f, and Cas12j nucleases along with their guide RNAs using a broad-host-range geminivirus, Wheat dwarf India virus (WDIV), in combination with Ageratum yellow leaf curl betasatellite (AYLCB). Cas9, Cas12f, and Cas12j nucleases were efficiently expressed along with corresponding guide RNAs under viral promoters. By leveraging tRNA spacers in place of external promoters and terminators, we significantly reduced the overall cargo size, streamlining vector design. Additionally, we compared the traditional AtU6-driven gRNA delivery with a novel spacer:gRNA:spacer format in Cas9-expressing lines and observed comparable editing efficiencies. The broad host range of WDIV and AYLCB, combined with this tissue culture-free genome editing platform, opens up possibilities for editing across a wide range of plant species.

plant biology↗

bioRxiv2025

Development of male-sterile lines of Setaria viridis to accelerate C4 model plant genetics

Setaria viridis is a diploid C4 grass in the Poaceae family, notable for its rapid life cycle of 6-8 weeks from sowing to seed--much shorter than the 4-5 months required by crops such as Zea mays and Sorghum bicolor. This fast growth makes S. viridis a valuable model for C4 crop research. Genetic crosses are essential for studying gene function, but manual crossing is labor-intensive and time-consuming. To address this, we developed a male-sterile line by targeting the S. viridis ortholog of Setaria italica NO POLLEN 1 (SiNP1), which encodes a glucose-methanol-choline oxidoreductase required for pollen exine formation. Using Cas9 and TREX2-mediated genome editing, we generated SiNP1 knockouts in both the S. viridis ME034V and A10.1 backgrounds that were fully male-sterile. Backcrossing T0 male-sterile plants to ME034V yielded a stable line homozygous for a 59 bp deletion, easily genotyped by PCR. Using this line, we developed a simple and efficient crossing protocol that eliminates the need for emasculation. This method enables a single person to perform up to 100 crosses per day--compared to 15 using traditional methods--and yields 20-32 F1 hybrid seeds per panicle with 100% genetic purity. We also quantified pollen flow and outcrossing frequencies under greenhouse conditions to develop optimal bagging strategies and prevent unintended pollination.This resource accelerates genetic research in S. viridis, enhancing its utility as a premier C4 model for mapping and functional genomics.

plant biology↗

bioRxiv2024

Viral delivery of recombinases to activate heritable genetic switches in plants

Viral vectors provide an increasingly versatile platform for transformation-free reagent delivery to plants. RNA viral vectors can be used to induce gene silencing, overexpress proteins, or introduce gene editing reagents, but they are often constrained by carrying capacity or restricted tropism in germline cells. Site-specific recombinases that catalyze precise genetic rearrangements are powerful tools for genome engineering that vary in size and, potentially, efficacy in plants. In this work, we show that viral vectors based on Tobacco rattle virus (TRV) deliver and stably express four recombinases ranging in size from [~]0.6kb to [~]1.5kb, and achieve simultaneous marker removal and reporter activation through targeted excision in transgenic Nicotiana benthamiana target lines. TRV vectors with Cre, FLP, CinH, and Integrase13 efficiently mediated recombination in infected somatic tissue, and also led to heritable modifications at high frequency. An excision-activated Ruby reporter enabled simple and high-resolution tracing of infected cell lineages, without the need for molecular genotyping. Together, our experiments broaden the scope of viral recombinase delivery, and offer insights into infection dynamics that may be useful in the development of future viral vectors.

plant biology↗

bioRxiv2022

Defining the Parameters to Improve Plant Regeneration with Developmental Regulators

Tissue culture methods which serve as the standard to regenerate modified plants are challenging and have limited the capacity to engineer new accessions. To improve upon these techniques, genome modifying reagents have been combined with developmental regulators to create gene edited plant tissues in both monocot and eudicot species. Co-culturing seedlings with Agrobacterium strains encoding developmental regulatory genes has proved to be effective at producing de novo meristems in multiple eudicot species. In order to see that this technology scales well beyond proof of concept experiments, various parameters were tested for refinement. Improvements have been observed at the key stages of growth induction and progression to shooting by manipulating the vector design, developmental regulator choice, Agrobacterium strain selection, and regulatory gene removal systems. Having defined these parameters as viable optimization points, the avenues to apply developmental regulators for plant regeneration in more diverse species have become more feasible.

plant biology↗

bioRxiv2022

Optimized dCas9 Transcription Activators for Plants

Understanding how gene expression impacts plant development and physiology is important for rationally engineering improved crops. Programmable Transcriptional Activators (PTAs), including CRISPR-Cas activators, have traditionally relied on a limited number of transcriptional activation domains. Usually the VP64 domain, derived from human herpes simplex virus, is fused to a DNA-binding domain to activate target gene expression. We reasoned there was considerable space for PTA improvement by replacing VP64 with a plant-derived activation domain. To address this, we designed, built, and tested a PTA library of 38 putative plant transcriptional activation domains. Domains from HSFA6b, AvrXa10, DOF1, DREB1, and DREB2 genes function as strong activators in Setaria viridis and Arabidopsis thaliana in protoplast assays and in transgenic plants. Overexpression of multiple endogenous genes (FT, PAP1, WUS) reached levels similar to the highly expressed housekeeping gene, PP2A, regardless of basal expression level. Further, these domains were effective in different PTA architectures, including the dCas9-SunTag, dCas9-Moontag, and TALE-SunTag systems. Lastly, we demonstrate the ability of these improved PTAs to map enhancer regions that promote gene expression in plants. This work demonstrates the effective and flexible nature of PTAs to activate target genes in plants, providing tools that can be used to improve agronomically relevant traits of interest.

plant biology↗

bioRxiv2022

High efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus

Delivery of gene editing components such as the Cas nuclease and single guide RNAs (sgRNAs) into plant cells is commonly accomplished by agrobacterium-mediated transformation. Although Arabidopsis is easy to transform, generation of biallelic edited plants requires screening a large number of plants in subsequent generations. Here, we describe optimization of the Tobacco rattle virus (TRV) for in planta delivery of sgRNAs fused to a tRNAIleu that induces efficient multiplex somatic and biallelic heritable editing in Arabidopsis. Inclusion of tRNAIleu enhances the systemic movement of TRV and the mutant phenotype is visible in the initial TRV::sgRNA-tRNAIleu infected Arabidopsis, which allows for the uncovering of lethal phenotypes. Mutant progeny are recovered in the next generation (M1) at frequencies ranging from 30-60%, with 100% mutant recovery in the following (M2) generation. TRV::tRNAIleu system described here allows generation of biallelic edited plants in a single generation and is amenable for large-scale high throughput CRISPR screens.

plant biology↗

bioRxiv2020

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis

In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed CRISPR/Cas9 system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples with 94% of deletions larger than 10bp, and less than 1% of mutations being insertions. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical NHEJ DNA repair pathway. Combined with the robust agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at the frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable MMEJ-mediated mutations in many plant species.

plant biology↗

bioRxiv2019

Building customizable auto-luminescent luciferase-based reporters in plants

Bioluminescence is a powerful biological signal that scientists have repurposed to design reporters for gene expression in plants and animals. However, there are some downsides associated with the need to provide a substrate to these reporters, such as its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP) in planta using an easily composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP in planta to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for the future construction of programmable auto-luminescent plant traits, such as creating light driven plant-pollinator interactions or light emitting plant-based sensors.

plant biology↗