Genome-edited Plants Research Articles

An accelerated transgene-free genome editing system using microparticle bombardment of sorghum immature embryos

Abstract The key factors for genome-editing in plants using CRISPR/Cas9, such as the Cas9 nuclease and guide RNA (gRNA) are typically expressed from a construct that is integrated into the plant genome. However, the presence of foreign DNA in the host genome causes genetic and regulatory concerns, particularly for commercialization. To address this issue, we developed an accelerated pipeline for generating transgene-free genome-edited sorghum (Sorghum bicolor) in the T0 generation. For proof-of-concept, we selected the Phytoene desaturase (PDS) gene as the target due to its visible phenotype (albinism) upon mutation. Following microprojectile-mediated co-transformation with a maize (Zea mays)-optimized Cas9 vector and a guide RNA (gRNA) cassette with a geneticin (G418) resistance gene, we divided tissue derived from immature embryos into two groups (with and without antibiotic selection) and cultured them separately as parallel experiments. In regenerated plants cultured on medium containing MS basal nutrition (to allow albino plants to survive), we detected higher rates of albinism in the non-selection group, achieving editing rates of 11.1–14.3% compared with 4.2–8.3% in the antibiotic selection group. In the T0 generation, 22.2–38.1% of albino plants from the non-selection group were identified as transgene-free, whereas only 0–5.9% from the selection group were transgene-free. Therefore, our strategy efficiently produced transgene-free genome-edited plants without the need for self-crossing or outcrossing, demonstrating the feasibility of achieving transgene-free genome-edited sorghum plants within a single generation. These findings pave the way for commercializing transgene-free genome-edited lines, particularly for vegetatively propagated crops like pineapple, sugarcane, and banana.

Harnessing promoter elements to enhance gene editing in plants: perspectives and advances.

Genome-edited plants, endowed with climate-smart traits, have been promoted as tools for strengthening resilience against climate change. Successful plant gene editing (GE) requires precise regulation of the GE machinery, a process controlled by the promoters, which drives its transcription through interactions with transcription factors (TFs) and RNA polymerase. While constitutive promoters are extensively used in GE constructs, their limitations highlight the need for alternative approaches. This review emphasizes the promise of tissue/organ specific as well as inducible promoters, which enable targeted GE in a spatiotemporal manner with no effects on other tissues. Advances in synthetic biology have paved the way for the creation of synthetic promoters, offering refined control over gene expression and augmenting the potential of plant GE. The integration of these novel promoters with synthetic systems presents significant opportunities for precise and conditional genome editing. Moreover, the advent of bioinformatic tools and artificial intelligence is revolutionizing the characterization of regulatory elements, enhancing our understanding of their roles in plants. Thus, this review provides novel insights into the strategic use of promoters and promoter editing to enhance the precision, efficiency and specificity of plant GE, setting the stage for innovative crop improvement strategies.

Rapid and Specific Colorimetric Assay for Detecting CRISPR-Cas9 in Genome-Edited Plants Using Loop-Mediated Isothermal Amplification

Rapid and Specific Colorimetric Assay for Detecting CRISPR-Cas9 in Genome-Edited Plants Using Loop-Mediated Isothermal Amplification

The Global Advance of Genome-Edited Plants to the Market: The Key Role of Chile in Its Development.

The global advancement of genome-edited plants toward commercialization has been significantly shaped by the functionality and flexibility of some regulatory frameworks governing plant genome editing. These frameworks vary widely across countries, reflecting diverse approaches to assessing and managing the risks and benefits of genome-editing technologies. While some nations have adopted product-based frameworks that focus on the characteristics of the final plant rather than the technique used, others rely on more restrictive process-based regulations. This variability influences the pace of innovation, the types of products able to enter the market, and their global trade potential. Chile stands out as a leader in this landscape, having implemented a science-driven and flexible regulatory framework. Its system promotes innovation by facilitating genome-edited plant research and development, field testing, and local commercialization. This regulatory adaptability positions Chile as a critical player in supporting the global integration of genome-editing technologies into agriculture, fostering advancements that address food security, sustainability, and climate resilience.

CRISPR-Cas9 Mediated Gene Editing Platform Through Callus-to-Plant Regeneration and Functional Analysis of DoALA4─DoALA6 in Dendrobium officinale.

Dendrobium orchids are well known for their great horticultural and medicinal values; however, the CRISPR/Cas9 gene editing system for Dendrobium species still needs to be improved. Therefore, this study aims to establish a CRISPR/Cas9-based functional validation system using Dendrobium officinale as a model species for the Dendrobium genus and to validate the DoALA4─DoALA6 genes, which may relate to growth and disease resistance. We first conducted a bioinformatics analysis of the P-type ATPase gene family in D. officinale, revealing the evolutionary diversity of P-type ATPase genes in orchids. Second, we inserted the GFP gene into the vector of CRISPR/Cas9 gene editing system to enhance the selection efficiency of genome-edited plants. Comparative analysis showed that different explants exhibited varying transformation efficiencies, ranging from 5% to 46.2%. Considering the regeneration capability, survival rate and gene editing efficiency, we selected callus as the transformation explant. Third, we used this editing system to generate DoALA4─DoALA6 mutants. Phenotypic observations of the mutants and inoculation of D. officinale with Sclerotium rolfsii indicated that DoALA4─DoALA6 are crucial for the growth of D. officinale and its resistance to southern blight disease. This efficient and stable CRISPR/Cas9 platform offers a foundation for further gene studies and Dendrobium breeding.

Jasmonate-mediated polyamine oxidase 6 drives herbivore-induced polyamine catabolism in rice.

Polyamines (PAs) along with their conjugated forms, are important mediators of plant defense mechanisms against both biotic and abiotic stresses. Flavin-containing polyamine oxidases (PAOs) regulate PA levels through terminal oxidation. To date, the role of PAOs in plant-herbivore interaction remains poorly understood. We discovered that infestation by the brown planthopper (BPH) disrupts PA homeostasis within the leaf sheaths of rice plants, which co-occurs with the upregulation of OsPAO6, a tissue-specific inducible, apoplast-localized enzyme that regulates the terminal catabolism of spermidine (Spd) and spermine. Functional analysis using CRISPR-Cas9 genome-edited plants revealed that pao6 mutants accumulated significantly higher levels of Spd and phenylpropanoid-conjugated Spd in response to BPH infestation compared to wild-type controls. In addition, BPH feeding on pao6 mutants led to increased honeydew excretion and plant damage by female adults, consistent with in vitro experiments in which Spd enhanced BPH feeding. Furthermore, OsPAO6 transcription is regulated by jasmonate (JA) signaling, and it is dependent on MYC2, which directly binds to the G-box-like motif in the OsPAO6 promoter. Our findings reveal an important role of OsPAO6 in regulating polyamine catabolism in JA-induced responses triggered by herbivore attacks in rice.

A DNA-free and genotype-independent CRISPR/Cas9 system in soybean.

Here, we report a smart genome editing system for soybean (Glycine max) using the in planta bombardment-ribonucleoprotein (iPB-RNP) method without introducing foreign DNA or requiring traditional tissue culture processes such as embryogenesis and organogenesis. Shoot apical meristem (SAM) of embryonic axes was used as the target tissue for genome editing because the SAM in soybean mature seeds has stem cells and specific cell layers that develop germ cells during the reproductive growth stage. In the iPB-RNP method, the RNP complex of the CRISPR/Cas9 system was directly delivered into SAM stem cells via particle bombardment, and genome-edited plants were generated from these SAMs. Soybean allergenic gene Gly m Bd 30K was targeted in this study. Many E0 (the first generation of genome-edited) plants in this experiment harbored mutant alleles at the targeted locus. Editing frequency of inducing mutations transmissible to the E1 generation was approximately 0.4% to 4.6% of all E0 plants utilized in various soybean varieties. Furthermore, simultaneous mutagenesis by iPB-RNP method was also successfully performed at other loci. Our results offer a practical approach for both plant regeneration and DNA-free genome editing achieved by delivering RNP into the SAM of dicotyledonous plants.

¿Podrían las nuevas propuestas regulatorias de la UE en materia de fitomejoramiento comprometer la innovación? Entre las expectativas generadas por la propuesta sobre Nuevas Técnicas Genómicas y el sinsentido de la propuesta sobre Material de Reproducción

The European Commission new regulatory proposals on new genome-edited plants and on plant reproductive material are set to revolutionize plant breeding and agriculture in the EU. This short communication analyzes the main features of such proposals with respect to the current regulatory frameworks in the EU, and discusses their foreseeable impact on European plant breeding innovation and agriculture. While there is no denying the potential advantages that the proposal on new genome-edited plants might bring, the proposed regulation is also complex and ambiguous, a matter already acknowledged by the specialized literature, and about which even some of the strongest advocates of the proposed regulation appear to show a certain degree of concern. If this is not corrected, the successful implementation of the proposal could be compromised. The proposal on plant reproductive material threatens to create new and probably unnecessary burdens for European plant breeders, farmers, and end consumers. Addressing the identified issues in the proposal on genome-edited plants, and drafting a new proposal on plant reproductive material aiming at reducing inefficiencies is suggested. Received: 21.12.2023Accepted: 10.05.2024

Characterization of organelle DNA degradation mediated by DPD1 exonuclease in the rice genome-edited line

Mitochondria and plastids, originated as ancestral endosymbiotic bacteria, contain their own DNA sequences. These organelle DNAs (orgDNAs) are, despite the limited genetic information they contain, an indispensable part of the genetic systems but exist as multiple copies, making up a substantial amount of total cellular DNA. Given this abundance, orgDNA is known to undergo tissue-specific degradation in plants. Previous studies have shown that the exonuclease DPD1, conserved among seed plants, degrades orgDNAs during pollen maturation and leaf senescence in Arabidopsis. However, tissue-specific orgDNA degradation was shown to differ among species. To extend our knowledge, we characterized DPD1 in rice in this study. We created a genome-edited (GE) mutant in which OsDPD1 and OsDPD1-like were inactivated. Characterization of this GE plant demonstrated that DPD1 was involved in pollen orgDNA degradation, whereas it had no significant effect on orgDNA degradation during leaf senescence. Comparison of transcriptomes from wild-type and GE plants with different phosphate supply levels indicated that orgDNA had little impact on the phosphate starvation response, but instead had a global impact in plant growth. In fact, the GE plant showed lower fitness with reduced grain filling rate and grain weight in natural light conditions. Taken together, the presented data reinforce the important physiological roles of orgDNA degradation mediated by DPD1.

Boosting genome editing in plants with single transcript unit surrogate reporter systems

Boosting genome editing in plants with single transcript unit surrogate reporter systems

CRISPR-Cas9 and beyond: identifying target genes for developing disease-resistant plants.

Throughout the history of crop domestication, desirable traits have been selected in agricultural products. However, such selection often leads to crops and vegetables with weaker vitality and viability than their wild ancestors when exposed to adverse environmental conditions. Considering the increasing human population and climate change challenges, it is crucial to enhance crop quality and quantity. Accordingly, the identification and utilization of diverse genetic resources are imperative for developing disease-resistant plants that can withstand unexpected epidemics of plant diseases. In this review, we provide a brief overview of recent progress in genome-editing technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) technologies. In particular, we classify disease-resistant mutants of Arabidopsis thaliana and several crop plants based on the roles or functions of the mutated genes in plant immunity and suggest potential target genes for molecular breeding of genome-edited disease-resistant plants. Genome-editing technologies are resilient tools for sustainable development and promising solutions for coping with climate change and population increases.

CRISPR/Cas9 ribonucleoprotein mediated DNA-free genome editing in larch.

Here, a DNA-free genetic editing approach is presented for larch by delivering ribonucleoprotein complexes (RNPs) of CRISPR/Cas9 through particle bombardment. The detailed procedure encompasses creating a transgenic system via particle bombardment for the transformation of embryogenic callus, validating the functionality of RNPs, optimizing coating and delivery techniques, enhancing somatic embryo maturation, regenerating plantlets, and precisely identifying mutants. The optimal particle bombardment parameters were determined at 1,100 psi and a distance of 9 cm and the editing efficiency of the targets was verified in vitro. Subsequently, the RNPs were transferred into the embryogenic callus. Mutant plants were obtained in targets 1 and target 2. The efficiencies of obtaining albino somatic embryos were 1.423% and 2.136%, respectively. A DNA-free particle bombardment transformation method suitable for larch has been established. The present study demonstrates that the DNA-free editing technology has been successfully implemented in larch. This method can achieve targeted genome editing in the larch genome, avoiding the risks of genomic integration and the lengthy breeding cycles associated with traditional transgenic methods. Moreover, it may be widely applicable for producing genome-edited conifer plants and holds great promise for commercialization.

An efficient protoplast-based genome editing protocol for Vitis species.

CRISPR-Cas technologies allow for precise modifications in plant genomes and promise to revolutionize agriculture. These technologies depend on the delivery of editing components into plant cells and the regeneration of fully edited plants. In vegetatively propagated plants, such as grape, protoplast culture provides one of the best avenues for producing non-chimeric and transgene-free genome-edited plants. However, poor regeneration of plants from protoplasts has hindered their implementation for genome editing. Here, we report an efficient protocol for regenerating plants from protoplasts from multiple grape varieties. By encapsulating the protoplasts in calcium alginate beads and co-culturing them with feeder cultures, the protoplasts divide to form callus colonies that regenerate into embryos and ultimately plants. This protocol worked successfully in wine and table grape (Vitis vinifera) varieties, as well as grape rootstocks and the grapevine wild relative Vitis arizonica. Moreover, by transfecting protoplasts with CRISPR-plasmid or ribonucleoprotein (RNP) complexes, we regenerated albino plants with edits in VvPHYTOENE DESATURASE gene in three varieties and in V. arizonica. The results reveal the potential of this platform to facilitate genome editing in Vitis species.

Study of functional features of plant root systems using CRISPR/Cas-mediated genome editing

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function [1, 2].
 A common construct for efficient genome editing and selection of hairy roots is comprised of three components, i.e., a cassette carrying the gene encoding the Cas nuclease, a cassette expressing the guide RNA (gRNA), and a cassette encoding a screenable or selectable marker [2]. After design and construction, the resulting vector is used to transform plant using appropriateRhizobium rhizogenesstrain.
 Over 26 plant species have been used in experiments combining genome editing and hairy root transformation to date [2]. Possible applications of CRISPR/Cas9 genome editing using hairy root transformation include different directions like test the efficiency of the CRISPR/Cas9 genome editing; obtaining whole genome-edited plants regenerated from individual edited hairy roots; investigation of root development or root function, root nodule symbiosis, resistance to biotic or abiotic stresses, or metabolic engineering [2].
 The basic principles of plant CRISPR/Cas genome editing like the different components of CRISPR/Cas vectors, the types of Cas nuclease, design principles of gRNAs, as well as the possible applications of CRISPR/Cas genome editing in hairy roots will discuss. The application of this method for multigene editing strategy will also be demonstrated onDEEPER ROOTING1genes of cucumber.
 The study was supported by the Ministry of Science and Higher Education of the Russian Federation (Grant No. 075-15-2021-1056).

Using New Bioinformatics Strategies at the Design Stage of Genome-edited Plants (Review)

Using New Bioinformatics Strategies at the Design Stage of Genome-edited Plants (Review)

Development of virus-induced genome editing methods in Solanaceous crops.

Genome editing (GE) using CRISPR/Cas systems has revolutionized plant mutagenesis. However, conventional transgene-mediated GE methods have limitations due to the time-consuming generation of stable transgenic lines expressing the Cas9/single guide RNA (sgRNA) module through tissue cultures. Virus-induced genome editing (VIGE) systems have been successfully employed in model plants, such as Arabidopsis thaliana and Nicotiana spp. In this study, we developed two VIGE methods for Solanaceous plants. First, we used the tobacco rattle virus (TRV) vector to deliver sgRNAs into a transgenic tomato (Solanum lycopersicum) line of cultivar Micro-Tom expressing Cas9. Second, we devised a transgene-free GE method based on a potato virus X (PVX) vector to deliver Cas9 and sgRNAs. We designed and cloned sgRNAs targeting Phytoene desaturase in the VIGE vectors and determined optimal conditions for VIGE. We evaluated VIGE efficiency through deep sequencing of the target gene after viral vector inoculation, detecting 40.3% and 36.5% mutation rates for TRV- and PVX-mediated GE, respectively. To improve editing efficiency, we applied a 37°C heat treatment, which increased the editing efficiency by 33% to 46% and 56% to 76% for TRV- and PVX-mediated VIGE, respectively. To obtain edited plants, we subjected inoculated cotyledons to tissue culture, yielding successful editing events. We also demonstrated that PVX-mediated GE can be applied to other Solanaceous crops, such as potato (Solanum tuberosum) and eggplant (Solanum melongena). These simple and highly efficient VIGE methods have great potential for generating genome-edited plants in Solanaceous crops.

Using New Bioinformatics Strategies at the Design Stage of Genome-edited Plants

The identification of risks associated with novel agricultural products of plant origin obtained via genome editing is an important aspect of genetic engineering. An extensive discussion is currently ongoing worldwide to clarify the similarities and differences between the “old” risks of “classic” GM plants and the “new” ones associated with genome editing, the lack of existing methods for identification and assessment of new risks. We propose here the concept of “safe by design” as applied to protection that is a new interesting tool that introduces good known standards of safety into plant bioengineering. This approach states that design options are identified to minimize or prevent risks and off-target of genome editing at the concept stage. The correlation between experimentally determined and in silico predicted off-target gRNA activity is a major challenge in the CRISPR system application. Today the most studies are focused on efficiency of gRNA design, while we pay attention specifically to the bioinformatics search and study of potential promoters, as the potential risk associates with a possible unplanned change in the transcriptional activity of promoters. We conveyed these strategies in the form of a risk assessment framework for regulation of new genetic technologies.

Efficient double-flowered gentian plant production using the CRISPR/Cas9 system

Japanese cultivated gentians are highly valued ornamental flowers in Japan, but the flower shape is mostly limited to the single-flower type, unlike other flowers such as roses and carnations. To overcome this limitation, we used the CRISPR/Cas9 genome editing system to increase double-flowered genetic resources in gentians. Our approach targeted an AGAMOUS (AG) floral homeotic gene (AG1), which is responsible for the natural mutation that causes double flowers in gentians. We designed two targets in exon 1 of AG1 for genome editing and found that 9 of 12 herbicide-resistant shoots had biallelic mutations in the target regions of AG1. These nine lines all produced double flowers, with stamens converted into petaloid organs, similar to the natural mutant. We also analyzed the off-target effects of AG2, which is homologous to AG1, and found that such effects occurred in gentian genome editing but with low frequency. Furthermore, we successfully produced transgene-free genome-edited plants (null segregants) by crossing with wild-type pollen. F1 seedlings were subjected to PCR analysis to determine whether foreign DNA sequences, two partial regions of the CaMV35S promoter and Cas9 gene, were present in the genome. As a result, foreign genes were segregated at a 1 : 1 ratio, indicating successful null segregant production. Using PCR analysis, we confirmed that four representative null segregants did not contain transfer DNA. In summary, our study demonstrates that the CRISPR/Cas9 system can efficiently produce double-flowered gentians, and null segregants can also be obtained. These genome-edited plants are valuable genetic resources for future gentian breeding programs.

Recalcitrance to transformation, a hindrance for genome editing of legumes.

Plant genome editing, a recently discovered method for targeted mutagenesis, has emerged as a promising tool for crop improvement and gene function research. Many genome-edited plants, such as rice, wheat, and tomato, have emerged over the last decade. As the preliminary steps in the procedure for genome editing involve genetic transformation, amenability to genome editing depends on the efficiency of genetic engineering. Hence, there are numerous reports on the aforementioned crops because they are transformed with relative ease. Legume crops are rich in protein and, thus, are a favored source of plant proteins for the human diet in most countries. However, legume cultivation often succumbs to various biotic/abiotic threats, thereby leading to high yield loss. Furthermore, certain legumes like peanuts possess allergens, and these need to be eliminated as these deprive many people from gaining the benefits of such crops. Further genetic variations are limited in certain legumes. Genome editing has the potential to offer solutions to not only combat biotic/abiotic stress but also generate desirable knock-outs and genetic variants. However, excluding soybean, alfalfa, and Lotus japonicus, reports obtained on genome editing of other legume crops are less. This is because, excluding the aforementioned three legume crops, the transformation efficiency of most legumes is found to be very low. Obtaining a higher number of genome-edited events is desirable as it offers the option to genotypically/phenotypically select the best candidate, without the baggage of off-target mutations. Eliminating the barriers to genetic engineering would directly help in increasing genome-editing rates. Thus, this review aims to compare various legumes for their transformation, editing, and regeneration efficiencies and discusses various solutions available for increasing transformation and genome-editing rates in legumes.

GENOME EDITING FOR EARLY AND LATE FLOWERING IN PLANTS

The use of genome editing to change the blooming period of plants has emerged as a valuable approach in contemporary agricultural research. This chapter delves into the complex processes that control early and late flowering in plants and how genome editing techniques such as CRISPR-Cas9 have altered the field. The chapter begins with an overview of the genetic pathways and regulatory networks that determine flowering time and then dives into the vital functions of key genes such as FLOWERING LOCUS T (FT), CONSTANS (CO), and FLOWERING LOCUS C (FLC). The chapter then delves into the many genome editing methods used to modify blooming time, focusing on augmentation and delay. Researchers have improved agricultural productivity, stress tolerance, and adaptation to changing climatic conditions by targeting regulatory genes. Case studies show effective genome editing applications in various plant species, indicating the possibility of crop development with personalized flowering time alterations. The ethical concerns and potential ecological implications of genome-edited plants with changed flowering times are also discussed, highlighting the significance of responsible research and environmental risk assessment. Furthermore, the chapter investigates the challenges and potential paths in the realm of genome editing for modifying flowering times in plants. This includes a comprehensive review of techniques to achieve more precise genetic modifications, strategies for reducing unintended alterations, and establishing regulatory guidelines.