CRISPR-Cas systems and applications for crop bioengineering.

CRISPR-Cas technologies contribute to enhancing our understanding of plant gene functions, and to the precise breeding of crop traits. Here, we review the latest progress in plant genome editing, focusing on emerging CRISPR-Cas systems, DNA-free delivery methods, and advanced editing approaches. By illustrating CRISPR-Cas applications for improving crop performance and food quality, we highlight the potential of genome-edited crops to contribute to sustainable agriculture and food security.

Identification of potential auxin response candidate genes for soybean rapid canopy coverage through comparative evolution and expression analysis.

Introduction: Throughout domestication, crop plants have gone through strong genetic bottlenecks, dramatically reducing the genetic diversity in today's available germplasm. This has also reduced the diversity in traits necessary for breeders to develop improved varieties. Many strategies have been developed to improve both genetic and trait diversity in crops, from backcrossing with wild relatives, to chemical/radiation mutagenesis, to genetic engineering. However, even with recent advances in genetic engineering we still face the rate limiting step of identifying which genes and mutations we should target to generate diversity in specific traits. Methods: Here, we apply a comparative evolutionary approach, pairing phylogenetic and expression analyses to identify potential candidate genes for diversifying soybean (Glycine max) canopy cover development via the nuclear auxin signaling gene families, while minimizing pleiotropic effects in other tissues. In soybean, rapid canopy cover development is correlated with yield and also suppresses weeds in organic cultivation. Results and discussion: We identified genes most specifically expressed during early canopy development from the TIR1/AFB auxin receptor, Aux/IAA auxin co-receptor, and ARF auxin response factor gene families in soybean, using principal component analysis. We defined Arabidopsis thaliana and model legume species orthologs for each soybean gene in these families allowing us to speculate potential soybean phenotypes based on well-characterized mutants in these model species. In future work, we aim to connect genetic and functional diversity in these candidate genes with phenotypic diversity in planta allowing for improvements in soybean rapid canopy cover, yield, and weed suppression. Further development of this and similar algorithms for defining and quantifying tissue- and phenotype-specificity in gene expression may allow expansion of diversity in valuable phenotypes in important crops.

Potato: from functional genomics to genetic improvement.

Potato is the most widely grown non-grain crop and ranks as the third most significant global food crop following rice and wheat. Despite its long history of cultivation over vast areas, slow breeding progress and environmental stress have led to a scarcity of high-yielding potato varieties. Enhancing the quality and yield of potato tubers remains the ultimate objective of potato breeding. However, conventional breeding has faced challenges due to tetrasomic inheritance, high genomic heterozygosity, and inbreeding depression. Recent advancements in molecular biology and functional genomic studies of potato have provided valuable insights into the regulatory network of physiological processes and facilitated trait improvement. In this review, we present a summary of identified factors and genes governing potato growth and development, along with progress in potato genomics and the adoption of new breeding technologies for improvement. Additionally, we explore the opportunities and challenges in potato improvement, offering insights into future avenues for potato research.

Kiwifruit in the Omics Age: Advances in Genomics, Breeding, and Beyond.

The kiwifruit, Actinidia genus, has emerged as a nutritionally rich and economically significant crop with a history rooted in China. This review paper examines the global journey of the kiwifruit, its genetic diversity, and the role of advanced breeding techniques in its cultivation and improvement. The expansion of kiwifruit cultivation from China to New Zealand, Italy, Chile and beyond, driven by the development of new cultivars and improved agricultural practices, is discussed, highlighting the fruit's high content of vitamins C, E, and K. The genetic resources within the Actinidia genus are reviewed, with emphasis on the potential of this diversity in breeding programs. The review provides extensive coverage to the application of modern omics technologies, including genomics, transcriptomics, proteomics, and metabolomics, which have revolutionized the understanding of the biology of kiwifruit and facilitated targeted breeding efforts. It examines both conventional breeding methods and modern approaches, like marker-assisted selection, genomic selection, mutation breeding, and the potential of CRISPR-Cas9 technology for precise trait enhancement. Special attention is paid to interspecific hybridization and cisgenesis as strategies for incorporating beneficial traits and developing superior kiwifruit varieties. This comprehensive synthesis not only sheds light on the current state of kiwifruit research and breeding, but also outlines the future directions and challenges in the field, underscoring the importance of integrating traditional and omics-based approaches to meet the demands of a changing global climate and market preferences.

Improvement of qualitative and quantitative traits in cotton under normal and stressed environments using genomics and biotechnological tools: A review.

Due to the increasing demand for high-quality and high fiber-yielding cotton (Gossypium spp.), research into the development of stress-resilient cotton cultivars has acquired greater significance. Various biotic and abiotic stressors greatly affect cotton production and productivity, posing challenges to the future of the textile industry. Moreover, the content and quality of cottonseed oil can also potentially be influenced by future environmental conditions. Apart from conventional methods, genetic engineering has emerged as a potential tool to improve cotton fiber quality and productivity. Identification and modification of genome sequences and the expression levels of yield-related genes using genetic engineering approaches have enabled to increase both the quality and yields of cotton fiber and cottonseed oil. Herein, we evaluate the significance and molecular mechanisms associated with the regulation of cotton agronomic traits under both normal and stressful environmental conditions. In addition, the importance of gossypol, a toxic phenolic compound in cottonseed that can limit consumption by animals and humans, is reviewed and discussed.

Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12iMax.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12iMax, a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12iMax in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163, and MSTN via Cas12iMax in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12iMax for gene editing in livestock animals and demonstrated the potential application of Cas12iMax in the field of animal trait improvement for agricultural production.

CRISPR enables sustainable cereal production for a greener future.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most important tool for targeted genome editing in many plant and animal species over the past decade. The CRISPR/Cas9 technology has also sparked a flood of applications and technical advancements in genome editing in the key cereal crops, including rice, wheat, maize, and barley. Here, we review advanced uses of CRISPR/Cas9 and derived systems in genome editing of cereal crops to enhance a variety of agronomically important features. We also highlight new technological advances for delivering preassembled Cas9-gRNA ribonucleoprotein (RNP)-editing systems, multiplex editing, gain-of-function strategies, the use of artificial intelligence (AI)-based tools, and combining CRISPR with novel speed breeding (SB) and vernalization strategies.

Identification of potential Auxin Response Candidate genes for soybean rapid canopy coverage through comparative evolution and expression analysis.

Glycine max, soybean, is an abundantly cultivated crop worldwide. Efforts have been made over the past decades to improve soybean production in traditional and organic agriculture, driven by growing demand for soybean-based products. Rapid canopy cover development (RCC) increases soybean yields and suppresses early-season weeds. Genome-wide association studies have found natural variants associated with RCC, however causal mechanisms are unclear. Auxin modulates plant growth and development and has been implicated in RCC traits. Therefore, modulation of auxin regulatory genes may enhance RCC. Here, we focus on the use of genomic tools and existing datasets to identify auxin signaling pathway RCC candidate genes, using a comparative phylogenetics and expression analysis approach. We identified genes encoding 14 TIR1/AFB auxin receptors, 61 Aux/IAA auxin co-receptors and transcriptional co-repressors, and 55 ARF auxin response factors in the soybean genome. We used Bayesian phylogenetic inference to identify soybean orthologs of Arabidopsis thaliana genes, and defined an ortholog naming system for these genes. To further define potential auxin signaling candidate genes for RCC, we examined tissue-level expression of these genes in existing datasets and identified highly expressed auxin signaling genes in apical tissues early in development. We identified at least 4 TIR1/AFB, 8 Aux/IAA, and 8 ARF genes with highly specific expression in one or more RCC-associated tissues. We hypothesize that modulating the function of these genes through gene editing or traditional breeding will have the highest likelihood of affecting RCC while minimizing pleiotropic effects.

Establishment of targeted mutagenesis in soybean protoplasts using CRISPR/Cas9 RNP delivery via electro-transfection.

The soybean (Glycine max L.) is an important crop with high agronomic value. The improvement of agronomic traits through gene editing techniques has broad application prospects in soybean. The polyethylene glycol (PEG)-mediated cell transfection has been successfully used to deliver the CRISPR/Cas9-based ribonucleoprotein (RNP) into soybean protoplasts. However, several downstream analyses or further cell regeneration protocols might be hampered by PEG contamination within the samples. Here in this study, we attempted to transfect CRISPR/Cas9 RNPs into trifoliate leaf-derived soybean protoplasts using Neon electroporation to overcome the need for PEG transfection for the first time. We investigated different electroporation parameters including pulsing voltage (V), strength and duration of pulses regarding protoplast morphology, viability, and delivery of CRISPR/Cas9. Electroporation at various pulsing voltages with 3 pulses and 10 ms per pulse was found optimal for protoplast electro-transfection. Following electro-transfection at various pulsing voltages (500 V, 700 V, 1,000 V, and 1,300 V), intact protoplasts were observed at all treatments. However, the relative frequency of cell viability and initial cell divisions decreased with increasing voltages. Confocal laser scanning microscopy (CLSM) confirmed that the green fluorescent protein (GFP)-tagged Cas9 was successfully internalized into the protoplasts. Targeted deep sequencing results revealed that on-target insertion/deletion (InDel) frequencies were increased with increasing voltages in protoplasts electro-transfected with CRISPR/Cas9 RNPs targeting constitutive pathogen response 5 (CPR5). InDel patterns ranged from +1 bp to -6 bp at three different target sites in CPR5 locus with frequencies ranging from 3.8% to 8.1% following electro-transfection at 1,300 V and 2.1% to 3.8% for 700 V and 1,000 V, respectively. Taken together, our results demonstrate that the CRISPR/Cas9 RNP system can be delivered into soybean protoplasts by the Neon electroporation system for efficient and effective gene editing. The electro-transfection system developed in this study would also further facilitate and serve as an alternative delivery method for DNA-free genome editing of soybean and other related species for genetic screens and potential trait improvement.

Opportunities and Challenges of In Vitro Tissue Culture Systems in the Era of Crop Genome Editing.

Currently, the development of genome editing (GE) tools has provided a wide platform for targeted modification of plant genomes. However, the lack of versatile DNA delivery systems for a large variety of crop species has been the main bottleneck for improving crops with beneficial traits. Currently, the generation of plants with heritable mutations induced by GE tools mostly goes through tissue culture. Unfortunately, current tissue culture systems restrict successful results to only a limited number of plant species and genotypes. In order to release the full potential of the GE tools, procedures need to be species and genotype independent. This review provides an in-depth summary and insights into the various in vitro tissue culture systems used for GE in the economically important crops barley, wheat, rice, sorghum, soybean, maize, potatoes, cassava, and millet and uncovers new opportunities and challenges of already-established tissue culture platforms for GE in the crops.

Exploring the potential of CRISPR/Cas genome editing for vegetable crop improvement: An overview of challenges and approaches.

Vegetables provide many nutrients in the form of fiber, vitamins, and minerals, which make them an important part of our diet. Numerous biotic and abiotic stresses can affect crop growth, quality, and yield. Traditional and modern breeding strategies to improve plant traits are slow and resource intensive. Therefore, it is necessary to find new approaches for crop improvement. Clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (CRISPR/Cas9) is a genome editing tool that can be used to modify targeted genes for desirable traits with greater efficiency and accuracy. By using CRISPR/Cas9 editing to precisely mutate key genes, it is possible to rapidly generate new germplasm resources for the promotion of important agronomic traits. This is made possible by the availability of whole genome sequencing data and information on the function of genes responsible for important traits. In addition, CRISPR/Cas9 systems have revolutionized agriculture, making genome editing more versatile. Currently, genome editing of vegetable crops is limited to a few vegetable varieties (tomato, sweet potato, potato, carrot, squash, eggplant, etc.) due to lack of regeneration protocols and sufficient genome sequencing data. In this article, we summarize recent studies on the application of CRISPR/Cas9 in improving vegetable trait development and the potential for future improvement.

CRISPR-Based Genome Editing and Its Applications in Woody Plants.

CRISPR/Cas-based genome editing technology provides straightforward, proficient, and multifunctional ways for the site-directed modification of organism genomes and genes. The application of CRISPR-based technology in plants has a vast potential value in gene function research, germplasm innovation, and genetic improvement. The complexity of woody plants genome may pose significant challenges in the application and expansion of various new editing techniques, such as Cas9, 12, 13, and 14 effectors, base editing, particularly for timberland species with a long life span, huge genome, and ploidy. Therefore, many novel optimisms have been drawn to molecular breeding research based on woody plants. This review summarizes the recent development of CRISPR/Cas applications for essential traits, including wood properties, flowering, biological stress, abiotic stress, growth, and development in woody plants. We outlined the current problems and future development trends of this technology in germplasm and the improvement of products in woody plants.

CRISPR/Cas9 System: A Potential Tool for Genetic Improvement in Floricultural Crops.

Demand of flowers is increasing with time worldwide. Floriculture has become one of the most important commercial trades in agriculture. Although traditional breeding methods like hybridization and mutation breeding have contributed significantly to the development of important flower varieties, flower production and quality of flowers can be significantly improved by employing modern breeding approaches. Novel traits of significance have interest to consumers and producers, such as fragrance, new floral color, change in floral architecture and morphology, vase life, aroma, and resistance to biotic and abiotic stresses, have been introduced by genetic manipulation. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has recently emerged as a powerful genome-editing tool for accurately changing DNA sequences at specific locations. It provides excellent means of genetically improving floricultural crops. CRISPR/Cas system has been utilized in gene editing in horticultural cops. There are few reports on the utilization of the CRISPR/Cas9 system in flowers. The current review summarizes the research work done by employing the CRISPR/Cas9 system in floricultural crops including improvement in flowering traits such as color modification, prolonging the shelf life of flowers, flower initiation, and development, changes in color of ornamental foliage by genome editing. CRISPR/Cas9 gene editing could be useful in developing novel cultivars with higher fragrance and enhanced essential oil and many other useful traits. The present review also highlights the basic mechanism and key components involved in the CRISPR/Cas9 system.

Simultaneous Improvement of Grain Yield and Quality through Manipulating Two Type C G Protein Gamma Subunits in Rice.

Heterotrimeric G protein signaling is an evolutionarily conserved mechanism in diverse organisms that mediates intracellular responses to external stimuli. In rice, the G proteins are involved in the regulation of multiple important agronomic traits. In this paper, we present our finding that two type C G protein gamma subunits, DEP1 and GS3, antagonistically regulated grain yield and grain quality. The DEP1 gene editing we conducted, significantly increased the grain number per panicle but had a negative impact on taste value, texture properties, and chalkiness-related traits. The GS3 gene editing decreased grain number per panicle but significantly increased grain length. In addition, the GS3 gene-edited plants showed improved taste value, appearance, texture properties, and Rapid Visco Analyser (RVA) profiles. To combine the advantages of both gs3 and dep1, we conducted a molecular design breeding at the GS3 locus of a "super rice" variety, SN265, which has a truncated dep1 allele with erect panicle architecture, high-yield performance, and which is of mediocre eating quality. The elongated grain size of the sn265/gs3 gene-edited plants further increased the grain yield. More importantly, the texture properties and RVA profiles were significantly improved, and the taste quality was enhanced. Beyond showcasing the combined function of dep1 and gs3, this paper presents a strategy for the simultaneous improvement of rice grain yield and quality through manipulating two type C G protein gamma subunits in rice.

National genomic evaluation of Korean thoroughbreds through indirect racing phenotype.

OBJECTIVE: Thoroughbred horses have been bred exclusively for racing in England for a long time. Additionally, because horse racing is a global sport, a healthy leisure activity for ordinary citizens, and a high-value business, systematic racehorse breeding at the population level is a requirement for continuous industrial development. Therefore, we established genomic evaluation system (using prize money as horse racing traits) to produce spirited, agile, and strong racing horse population. METHODS: We used phenotypic data from 25,061 Thoroughbred horses (all registered individuals in Korea) that competed in races between 1994 and 2019 at the Korea Racing Authority and constructed pedigree structures. We quantified the improvement in racehorse breeding output by year in Korea, and this aided in the establishment of a high-level horse-fill industry. RESULTS: We found that pedigree-based best linear unbiased prediction method improved the racing performance of the Thoroughbred population with high accuracy, making it possible to construct an excellent Thoroughbred racehorse population in Korea. CONCLUSION: This study could be used to develop an efficient breeding program at the population level for Korean Thoroughbred racehorse populations as well as others.

Advances and Perspectives of Transgenic Technology and Biotechnological Application in Forest Trees.

Transgenic technology is increasingly used in forest-tree breeding to overcome the disadvantages of traditional breeding methods, such as a long breeding cycle, complex cultivation environment, and complicated procedures. By introducing exogenous DNA, genes tightly related or contributed to ideal traits-including insect, disease, and herbicide resistance-were transferred into diverse forest trees, and genetically modified (GM) trees including poplars were cultivated. It is beneficial to develop new varieties of GM trees of high quality and promote the genetic improvement of forests. However, the low transformation efficiency has hampered the cultivation of GM trees and the identification of the molecular genetic mechanism in forest trees compared to annual herbaceous plants such as Oryza sativa. In this study, we reviewed advances in transgenic technology of forest trees, including the principles, advantages and disadvantages of diverse genetic transformation methods, and their application for trait improvement. The review provides insight into the establishment and improvement of genetic transformation systems for forest tree species. Challenges and perspectives pertaining to the genetic transformation of forest trees are also discussed.

CRISPR Genome Editing Technology: A Powerful Tool Applied to Developing Agribusiness.

The natural increase of the world's population implies boosting agricultural demand. In the current non-optimistic global scenario, where adverse climate changes come associated with substantial population growth, the main challenge in agribusiness is food security. Recently, the CRISPR/Cas system has emerged as a friendly gene editing biotechnological tool, enabling a precise manipulation of genomes and enhancement of desirable traits in several organisms. This review highlights the CRISPR/Cas system as a paramount tool for the improvement of agribusiness products and brings up-to-date findings showing its potential applications in improving agricultural-related traits in major plant crops and farm animals, all representing economic-relevant commodities responsible for feeding the world. Several applied pieces of research have successfully demonstrated the CRISPR/Cas ability in boosting interesting traits in agribusiness products, including animal productivity and welfare, crop yield growth, and seed quality, reflecting positive impacts in both socioeconomics and human health aspects. Hence, the CRISPR/Cas system has revolutionized bioscience and biotechnology, and its concrete application in agribusiness goods is on the horizon.

CRISPR/Cas9-Mediated Generation of Pathogen-Resistant Tomato against Tomato Yellow Leaf Curl Virus and Powdery Mildew.

Tomato is one of the major vegetable crops consumed worldwide. Tomato yellow leaf curl virus (TYLCV) and fungal Oidium sp. are devastating pathogens causing yellow leaf curl disease and powdery mildew. Such viral and fungal pathogens reduce tomato crop yields and cause substantial economic losses every year. Several commercial tomato varieties include Ty-5 (SlPelo) and Mildew resistance locus o 1 (SlMlo1) locus that carries the susceptibility (S-gene) factors for TYLCV and powdery mildew, respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a valuable genome editing tool to develop disease-resistant crop varieties. In this regard, targeting susceptibility factors encoded by the host plant genome instead of the viral genome is a promising approach to achieve pathogen resistance without the need for stable inheritance of CRISPR components. In this study, the CRISPR/Cas9 system was employed to target the SlPelo and SlMlo1 for trait introgression in elite tomato cultivar BN-86 to confer host-mediated immunity against pathogens. SlPelo-knockout lines were successfully generated, carrying the biallelic indel mutations. The pathogen resistance assays in SlPelo mutant lines confirmed the suppressed accumulation of TYLCV and restricted the spread to non-inoculated plant parts. Generated knockout lines for the SlMlo1 showed complete resistance to powdery mildew fungus. Overall, our results demonstrate the efficiency of the CRISPR/Cas9 system to introduce targeted mutagenesis for the rapid development of pathogen-resistant varieties in tomato.

Understanding Filipino Rice Farmer Preference Heterogeneity for Varietal Trait Improvements: A Latent Class Analysis.

Using an experimental methodology based on investment games, we examine whether smallholder rice farmers from Nueva Ecija, Philippines have heterogeneous preferences for improvements in 10 rice varietal traits. We use a latent class cluster approach to identify different segments of rice producing households and their distinct preferences for trait improvements. These clusters were characterised post hoc using household, farm, and marketing characteristics. On average, farmers invested the most in rice varietal trait improvements that offered opportunities to reduce losses caused by lodging, insects and diseases. We found four classes of farmers with distinct preferences for improvements in variety traits. The clusters were significantly different in terms of household and farm characteristics. These findings can guide breeding research in the development of varieties that have the traits farmers identified for improvement, and that will address the unique needs of distinct farmer segments.