Genetic engineering of plants has enhanced crop productivity in the face of climate change and a growing global population by conferring desirable genetic traits to agricultural crops. Efficient ...genetic transformation in plants remains a challenge due to the cell wall, a barrier to exogenous biomolecule delivery. Conventional delivery methods are inefficient, damaging to tissue, or are only effective in a limited number of plant species. Nanoparticles are promising materials for biomolecule delivery, owing to their ability to traverse plant cell walls without external force and highly tunable physicochemical properties for diverse cargo conjugation and broad host range applicability. With the advent of engineered nuclease biotechnologies, we discuss the potential of nanoparticles as an optimal platform to deliver biomolecules to plants for genetic engineering.
Plant biotechnology is key to ensuring food and energy security; however, biomolecule delivery and progeny regeneration continue to be key challenges in plant genetic engineering.
Conventional biomolecule delivery methods in plants have critical drawbacks, such as low efficiency, narrow species range, limited cargo types, and tissue damage.
Advances in nanotechnology have created opportunities to overcome limitations in conventional methods: nanoparticles are promising for species-independent passive delivery of DNA, RNA, and proteins.
The advent of nuclease-based genome editing (e.g., CRISPR-Cas9) has ushered in a new era of precise genetic engineering that, among other impacts, has enabled the development of genetically engineered crops without harsh regulatory restrictions.
The potential of nanoparticles to overcome limitations in conventional delivery makes them excellent candidates for delivery of nuclease-based genome editing cargo, thus making nanoparticle delivery a critical technology for the advancement of plant genetic engineering.
Delivery of biomolecules to plants relies on Agrobacterium infection or biolistic particle delivery, the former of which is amenable only to DNA delivery. The difficulty in delivering functional ...biomolecules such as RNA to plant cells is due to the plant cell wall, which is absent in mammalian cells and poses the dominant physical barrier to biomolecule delivery in plants. DNA nanostructure-mediated biomolecule delivery is an effective strategy to deliver cargoes across the lipid bilayer of mammalian cells; however, nanoparticle-mediated delivery without external mechanical aid remains unexplored for biomolecule delivery across the cell wall in plants. Herein, we report a systematic assessment of different DNA nanostructures for their ability to internalize into cells of mature plants, deliver siRNAs, and effectively silence a constitutively expressed gene in Nicotiana benthamiana leaves. We show that nanostructure internalization into plant cells and corresponding gene silencing efficiency depends on the DNA nanostructure size, shape, compactness, stiffness, and location of the siRNA attachment locus on the nanostructure. We further confirm that the internalization efficiency of DNA nanostructures correlates with their respective gene silencing efficiencies but that the endogenous gene silencing pathway depends on the siRNA attachment locus. Our work establishes the feasibility of biomolecule delivery to plants with DNA nanostructures and both details the design parameters of importance for plant cell internalization and also assesses the impact of DNA nanostructure geometry for gene silencing mechanisms.
Many efforts have been put into engineering plants to improve crop yields and stress tolerance and boost the bioproduction of valuable molecules. Yet, our capabilities are still limited due to the ...lack of well-characterized genetic building blocks and resources for precise manipulation and given the inherently challenging properties of plant tissues. Advancements in plant synthetic biology can overcome these bottlenecks and release the full potential of engineered plants. In this review, we first discuss the recently developed plant synthetic elements from single parts to advanced circuits, software, and hardware tools expediting the engineering cycle. Next, we survey the advancements in plant biotechnology enabled by these recent resources. We conclude the review with outstanding challenges and future directions of plant synthetic biology.
Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis and crop engineering. The plant cell wall is a barrier that limits the ease and throughput of ...exogenous biomolecule delivery to plants. Current delivery methods either suffer from host-range limitations, low transformation efficiencies, tissue damage or unavoidable DNA integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into intact plants of several species with pristine and chemically functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene integration, into plant cells for diverse biotechnology applications.
CRISPR-Cas genetic engineering of plants holds tremendous potential for providing food security, battling biotic and abiotic crop stresses caused by climate change, and for environmental remediation ...and sustainability. Since the discovery of CRISPR-Cas technology, its usefulness has been demonstrated widely, including for genome editing in plants. Despite the revolutionary nature of genome-editing tools and the notable progress that these tools have enabled in plant genetic engineering, there remain many challenges for CRISPR applications in plant biotechnology. Nanomaterials could address some of the most critical challenges of CRISPR genome editing in plants through improvements in cargo delivery, species independence, germline transformation and gene editing efficiency. This Perspective identifies major barriers preventing CRISPR-mediated plant genetic engineering from reaching its full potential, and discusses ways that nanoparticle technologies can lower or eliminate these barriers. We also describe advances that are needed in nanotechnology to facilitate and accelerate plant genome editing. Timely advancement of the application of CRISPR technologies in plant engineering is crucial for our ability to feed and sustain the growing human population under a changing global climate.
Exogenous biomolecule delivery into plants is difficult because the plant cell wall poses a dominant transport barrier, thereby limiting the efficiency of plant genetic engineering. Traditional DNA ...delivery methods for plants suffer from host-species limitations, low transformation efficiencies, tissue damage, or unavoidable and uncontrolled DNA integration into the host genome. We have demonstrated efficient plasmid DNA delivery into intact plants of several species with functionalized high-aspect-ratio carbon nanotube (CNT) nanoparticles (NPs), enabling efficient DNA delivery into a variety of non-model plant species (arugula, wheat, and cotton) and resulting in high protein expression levels without transgene integration. Herein, we provide a protocol that can be implemented by plant biologists and adapted to produce functionalized single-walled CNTs (SWNTs) with surface chemistries optimized for delivery of plasmid DNA in a plant species-independent manner. This protocol describes how to prepare, construct, and optimize polyethylenimine (PEI)-functionalized SWNTs and perform plasmid DNA loading. The authors also provide guidance on material characterization, gene expression evaluation, and storage conditions. The entire protocol, from the covalent functionalization of SWNTs to expression quantification, can be completed in 5 d.
Food security is threatened by rising global population and effects of climate change. Most of our calories come from a few crops that are difficult to improve. Lowe et al. developed a plant ...transformation approach enabling crop genetic engineering that could provide a route to a future with greater food security.
RNA interference, which involves the delivery of small interfering RNA (siRNA), has been used to validate target genes, to understand and control cellular metabolic pathways, and to use as a “green” ...alternative to confer pest tolerance in crops. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit plant species range limitations and uncontrolled DNA integration into the plant genome. Here, we synthesize polyethylenimine-functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plants and show that these nanoclusters enable efficient gene knockdown. We further demonstrate that PEI-AuNCs protect siRNA from RNase degradation while the complex is small enough to bypass the plant cell wall. Consequently, AuNCs enable gene knockdown with efficiencies of up 76.5 ± 5.9% and 76.1 ± 9.5% for GFP and ROQ1, respectively, with no observable toxicity. Our data suggest that AuNCs can deliver siRNA into intact plant cells for broad applications in plant biotechnology.
Engineering plant microbiomes presents a promising strategy for advancing sustainable agriculture practices.Genome-wide association studies can identify plant host genomic regions that influence ...microbiome structure and function, thereby informing genetic engineering and targeted breeding strategies.Co-inoculation experiments and metabolic modeling can identify critical microbial interspecies interactions.The incorporation of machine learning algorithms into microbiome research has expanded the predictive capabilities.Integration of experimental and computational tools has been successful in designing microbiomes with precise functions.
Plant growth-promoting rhizobacteria (PGPR) can improve crop yields, nutrient use efficiency, plant tolerance to stressors, and confer benefits to future generations of crops grown in the same soil. Unlocking the potential of microbial communities in the rhizosphere and endosphere is therefore of great interest for sustainable agriculture advancements. Before plant microbiomes can be engineered to confer desirable phenotypic effects on their plant hosts, a deeper understanding of the interacting factors influencing rhizosphere community structure and function is needed. Dealing with this complexity is becoming more feasible using computational approaches. In this review, we discuss recent advances at the intersection of experimental and computational strategies for the investigation of plant–microbiome interactions and the engineering of desirable soil microbiomes.
Plant growth-promoting rhizobacteria (PGPR) can improve crop yields, nutrient use efficiency, plant tolerance to stressors, and confer benefits to future generations of crops grown in the same soil. Unlocking the potential of microbial communities in the rhizosphere and endosphere is therefore of great interest for sustainable agriculture advancements. Before plant microbiomes can be engineered to confer desirable phenotypic effects on their plant hosts, a deeper understanding of the interacting factors influencing rhizosphere community structure and function is needed. Dealing with this complexity is becoming more feasible using computational approaches. In this review, we discuss recent advances at the intersection of experimental and computational strategies for the investigation of plant–microbiome interactions and the engineering of desirable soil microbiomes.
During the last couple of decades considerable research efforts have been directed towards the synthesis and coating of iron oxide nanoparticles (IONPs) for biomedical applications. To address the ...current limitations, recent studies have focused on the design of new generation nanoparticle systems whose internalization and targeting capabilities have been improved through surface modifications. This review covers the most recent challenges and advances in the development of IONPs with enhanced quality, and biocompatibility for various applications in biotechnology and medicine.
Synthesis and design of biologically inspired biocompatible iron oxide nanoparticles.
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