Biology subjects

Khakhar, A.

Publications and source records attributed to Khakhar, A..

10 recordsLinked to original sources

bioRxiv2026

Agrochemical-responsive gene expression control systems for modulating plant development and metabolism

Precise temporal control of gene expression is critical for studying plant biology and engineering complex crop traits. Current systems enable chemically inducible regulation but rely on costly or agriculturally impractical inducers and lack the flexibility needed to regulate combinations of native loci and transgenes. In this work, we elucidate the design rules for control systems, based on Cas9 and Cre recombinase fused to the ecdysone receptor (EcR), which respond to a widely used agrochemical methoxyfenozide (MF). First, we validated the function of both circuits in transient assays and explored how transduction properties can be modulated by engineering nuclear trafficking dynamics. We next characterized both the Cas9-based and recombinase-based systems by using them to regulate fluorescent reporters in transgenic Arabidopsis thaliana plants. Here, we demonstrate systemic activation following root application of MF, validating the use of an agriculturally compatible inducer for whole-plant gene regulation. Finally, we validated the utility of the inducible Cas-based SynTF system to regulate multigene pathways and control both metabolic flux and developmental circuits. Together, these results establish design principles for agrochemical-inducible control systems and demonstrate their utility for temporally regulating plant phenotypes. These synthetic circuits provide a versatile framework for engineering complex traits using an agriculturally compatible inducer.

synthetic biology↗

bioRxiv2026

High-throughput engineering of ligand-activated splicing ribozyme through domain insertion

Domain insertion is an established method to engineer ligand-mediated control of activity in protein scaffolds. Whether this strategy can be systematically applied to large, structured RNAs remains unclear. In this study, we investigated the feasibility of engineering ligand-activated splicing ribozymes (LASRs) from group I catalytic introns. Using domain-insertion profiling coupled with high-throughput screening, we mapped the nucleotide-resolution landscape of aptamer insertion across the ribozyme and identified sites that support robust ligand-dependent control. We showed LASRs function across multiple kingdoms of life, including diverse species of bacteria and even fungi, and can be used to regulate various genetic outputs. Finally, we integrated LASRs with a genetic recorder that writes information into ribosomal RNA, enabling sequencing-based recovery of intracellular chemical signals from microbial consortia. This work establishes LASRs as an RNA-based inducible control platform for sensing diverse chemical inputs, regulating the expression of diverse genes of interest, and recording intracellular information.

synthetic biology↗

bioRxiv2026

A CURE for synthetic regulation of gene expression: Rapid screening of guide RNA efficacy as a framework for enabling undergraduate research in plant synthetic biology

Course-based Undergraduate Research Experiences (CUREs) have emerged as a transformative approach to science education, expanding access to authentic research opportunities beyond the traditional undergraduate research assistant (URA) training. By embedding research into a curriculum, CUREs engage a broad and diverse population of students in a classroom environment that emphasizes experimental design, data analysis, and scientific communication. However, this has been difficult to develop for fields such as plant synthetic biology due to the long timescales of plant transformation. One avenue around this problem is to utilize a recent innovation that enables high throughput and rapid screening of gRNA efficacy by leveraging viral-based delivery of guide RNAs (gRNAs). In this work, we develop and validate a CURE with undergraduate students at Colorado State University (CSU). Students worked in teams to design and test efficacy of gRNAs targeting a Cas9-based transcriptional repressor to different regions of the promoters of the three GIBBERELLIN INSENSITIVE 1 genes (GID1a, GID1b, and GID1c) in Arabidopsis thaliana. Over the semester, students generated and analyzed gene expression data to understand the efficiency of twelve new gRNAs. We further validated CURE student-identified gRNAs with an undergraduate research assistant (URA) that assessed target gene expression and phenotypic outcomes in stable transgenic lines expressing SynTF constructs with the strongest gRNAs from the class. We further describe the curriculum structure to facilitate adoption at other institutions and present student-generated datasets demonstrating the utility of ViN-based screening for identifying effective SynTF gRNAs for plant functional genomics and engineering.

synthetic biology↗

bioRxiv2025

Uncovering the Design Rules for Sustainable Growth of Mineralized Mycomaterials

Mycomaterials, materials made from filamentous fungi, have several advantages over traditional materials, such as their genetic programmability and self-healing properties. However, their lack of mechanical strength and cost of production often constrain the applications they can be used in. In this work, we take inspiration from natural systems to overcome these challenges by elucidating design principles for mineralization-based enhancement of mechanical strength and synthetic lichen-based low-cost growth. We demonstrate that surface display of an enzyme from sea sponges, silicatein , on the hyphae of the filamentous fungus Aspergillus niger enables mineralization of polysilicate and that this does not impact fungal growth. We also show that this strategy can be extended to other silicatein variants and characterize how the degree of mineralization can be modulated. We then demonstrate that mineralization enhances the mechanical properties of the mycelium, including its tensile strength, modulus, and toughness. Finally, we show how these reinforced mycelia can be grown without external carbon sources using a synthetic lichen-based co-culture to facilitate low cost biomanufacturing. Together, our results lay the groundwork for the sustainable production of mineralized mycomaterials and create a new model system to study how mineralization impacts growth and mechanical properties. Significance StatementMaterials made from filamentous fungi, called mycomaterials, have several advantages over traditional materials, but their poor mechanical properties and relatively high production costs have limited their application. We elucidated design principles to enable tunable mineralization of fungal mycelium and have shown that it does not impact growth but significantly enhances mechanical strength. We have also shown that these reinforced mycelia can be grown without any external carbon in a synthetic lichen like consortia to minimize production costs. This work creates a novel experimental system to study how mineralization impacts growth and mechanical properties and will facilitate the broader application of mycomaterials in the future.

synthetic biology↗

bioRxiv2025

Elucidating design principles for Ribozyme-Enabled Tissue Specificity (RETS) to allow precise expression without specialized promoters.

Tissue- or cell type- specific expression of transgenes is often essential for interrogation of biological phenomenon or predictable engineering of multicellular organisms but can be stymied by cryptic enhancers that make identification of promoters that generate desired expression profiles challenging. In plants the months-to-years long timeline associated with prototyping putative tissue-specific promoters in transgenic lines deepens this challenge. We have developed a novel strategy called Ribozyme Enabled Tissue Specificity (RETS) that leverages the knowledge of where and when genes are expressed derived from transcriptomic studies to enable tissue-specific expression without needing characterized promoters. It uses a split self-splicing ribozyme based on a group I intron from Tetrahymena thermophila to enable conditional reconstitution of a transgene mRNA in the presence of a secondary tissue-specific mRNA of choice. We elucidate the design features that enable flexible swapping of transgenes and targets, enhancing transgene expression, and circumventing host RNA interference responses. We then show that these innovations enable tissue-specific and dose-dependent expression of transgenes in Arabidopsis thaliana. Finally, we demonstrate the utility of RETS both for creating genetically encoded biosensors to study the spatiotemporal patterns of gene expression in planta as well as for engineering tissue-specific changes in organ size. RETS provides a novel avenue to study expression patterns of native loci with non-destructive imaging, complementing the weakness of existing approaches. Additionally, the spatiotemporal control of transgene expression afforded by RETS enables precision engineering of plant phenotypes which will facilitate enhancing crops without the trade-offs associated with constitutive expression.

synthetic biology↗

bioRxiv2025

Molecular Bonsai: Elucidating the design principles for engineering plant organ size

Enhancements to crop morphology, such as the semi-dwarfing that helped drive the green revolution, are often driven by changes in gene expression. These are challenging to translate across species, which slows the rate of crop improvement. Synthetic transcription factors (SynTFs) offer a rapid alternative to generate targeted alterations to gene expression. However, the complexity of developmental pathways makes it unclear how to best apply them to predictably engineer morphology. In this work, we explore whether mathematical modeling can guide SynTF-based gene expression modulation to help elucidate the design principles of engineering organ size. We targeted genes in the phytohormone, gibberellin (GA), signaling pathway, which is a central regulator of cell expansion. We demonstrate that modulation of GA signaling gene expression can generate consistent dwarfing across tissues and environments in Arabidopsis thaliana, and that the degree of dwarfing is dependent on the strength of regulation, as predicted by modeling. We further validate the models predictive power by demonstrating its capacity to predict the qualitative impacts of different regulatory architectures for engineering organ size. Additionally, we develop expression parameterized models to quantitatively predict organ size and elucidate how temperature will affect growth. Finally, we show that these insights can be generalized for engineering organ size in tomato (Solanum lycopersicum). This work creates a framework for predictable engineering of an agriculturally important trait across tissues and plant species. It also serves as a proof-of-concept for how mathematical models can guide SynTF-based alterations in gene expression to enable bottom-up design of plant phenotypes. Significance StatementWhile traditional breeding approaches have identified mutations that enhance crop performance via targeted gene expression changes, these are not easily translated across varieties and species. Synthetic transcription factors (SynTFs) offer an avenue to generate such changes de novo, but the optimal regulatory architectures necessary to generate desired phenotypes remain unclear. We demonstrate how mathematical models can be used to guide SynTF deployment and elucidate the design principles for engineering organ size, an agriculturally important trait, via modulation of gibberellin signaling. In addition to revealing regulatory architectures that can consistently increase or decrease organ size across a range of tissues, environments, and plant species, this work demonstrates how model-guided SynTF-based modulation of gene dosage can be used to predictably engineer plants.

synthetic biology↗

bioRxiv2025

Reprogramming feedback strength in gibberellin biosynthesis highlights conditional regulation by the circadian clock and carbon dioxide.

The phytohormone gibberellin (GA) is an important regulator of plant morphology and reproduction, and the biosynthesis and distribution of GA in planta is agriculturally relevant to past and current breeding efforts. Tools like biosensors, extensive molecular genetic resources in reference plants and mathematical models have greatly contributed to current understanding of GA homeostasis; however, these tools are difficult to tune or repurpose for engineering crop plants. Previously, we showed that a GA-regulated Hormone Activated CAS9-based Repressor (GAHACR) functions in planta. Here, we use GAHACRs to modulate the strength of feedback on endemic GA regulated genes, and to directly test the importance of transcriptional feedback in GA signaling. We first adapted existing mathematical models to predict the impact of targeting a GAHACR to different nodes in the GA biosynthesis pathway, and then implemented a perturbation predicted by the model to lower GA levels. Specifically, we individually targeted either the biosynthetic gene GA20 oxidase (GA20ox) or the GA receptor GID1, and characterized primary root length, flowering time and the transcriptome of these transgenic lines. Using this approach, we identified a strong connection between GA signaling status and the circadian clock, which can be largely attenuated by elevated carbon dioxide levels. Our results identify a node in the GA signaling pathway that can be engineered to modulate plant size and flowering time. Our results also raise concerns that rising atmospheric CO2 concentration are likely to reverse many of the gains of Green Revolution crops.

synthetic biology↗

bioRxiv2020

VipariNama: RNA vectors to rapidly reprogram plant morphology and metabolism

Synthetic transcription factors have great promise as tools to explore biological processes. By allowing precise alterations in gene expression, they can help elucidate relationships between gene expression and plant morphology or metabolism. However, the years-long timescales, high cost, and technical skill associated with plant transformation have dramatically slowed their use. In this work, we developed a new platform technology called VipariNama (ViN) in which RNA vectors are used to rapidly deploy synthetic transcription factors and reprogram gene expression in planta. We demonstrate how ViN vectors can direct activation or repression of multiple genes, systemically and persistently over several weeks, and in multiple plant species. We also show how this transcriptional reprogramming can create predictable changes to metabolic and morphological phenotypes in the model plants Nicotiana benthamiana and Arabidopsis thaliana in a matter of weeks. Finally, we show how a model of gibberellin signaling can guide ViN vector-based reprogramming to rapidly engineer plant size in both model species as well as the crop Solanum lycopersicum (tomato). In summary, using VipariNama accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

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↗

bioRxiv2017

Synthetic hormone-responsive transcription factors can monitor and re-program plant development

Developmental programs continuously sculpt plant morphology to meet environmental challenges, and these same programs have been manipulated to increase agricultural productivity1,2. Small molecule phytohormones act as signals within these programs creating chemical circuitry3 that, in many cases, has been represented in mathematical models4,5. To date, model validation and forward engineering of plant morphology has been largely restricted to adding or subtracting genes, as more nuanced tools to modulate key control parameters identified by such models in vivo are severely limited6,7. Here, we use Arabidopsis thaliana to validate a novel set of synthetic and modular hormone activated Cas9-based repressors (HACRs) that respond to three phytohormones: auxin, gibberellins and jasmonates. We demonstrate that HACRs can regulate genes in response to both exogenous hormone treatments, as well as in response to local differences in endogenous hormone levels associated with developmental events. We further show that HACRs can be used to reprogram the agriculturally relevant traits of shoot branching and phyllotaxy by tuning canalization strength, a critical control parameter predicted by mathematical models. By deploying a HACR to re-parameterize the threshold for induction of the auxin transporter PIN-FORMED1 (PIN1), we observed a decrease in shoot branching and phyllotactic noise as predicted by existing models4,5. The approach described here provides a framework for improved mapping of developmental circuitry, as well as a means to better leverage model predictions to engineer development.

plant biology↗