Balancing Trait Improvement with Tradeoff Side-Effects Using Genome Editing Technology

Crop improvement and adaptation are critical to ensure global food security in the face of climate change, population growth, and resource limitations. Exploiting genomic resources and molecular breeding techniques offers immense potential to accelerate the development of resilient, high-yielding crop varieties. This review provides an overview of the current state and future prospects of leveraging genomics and molecular breeding for crop improvement, with a focus on major food crops. We discuss key genomic resources such as reference genomes, transcriptomes, and pan-genomes, as well as powerful molecular breeding approaches like marker-assisted selection, genomic selection, and genome editing. Integrating these tools into crop breeding pipelines can greatly enhance the precision and efficiency of developing improved varieties with desirable traits such as abiotic stress tolerance, disease resistance, and enhanced nutritional quality. We also highlight successful examples of applying these techniques in crops like rice, wheat, maize, and legumes. Furthermore, we explore the role of big data, machine learning, and systems biology in extracting actionable insights from the vast genomic data being generated. Finally, we discuss the challenges and opportunities in translating genomic discoveries into improved crop varieties, emphasizing the need for multidisciplinary collaborations, capacity building, and public-private partnerships. Harnessing the power of genomics and molecular breeding will be pivotal in developing climate-resilient crops to feed the growing global population sustainably.

In an era of climate change along with increasing world population, the food security can be ensured by developing climate resilient crop varieties having good nutritional quality. Improved crop varieties can be developed through importation of desirable features which encompass the discrimination of available variability through introduction and selection as primitive method. Conventional breeding methods take more time in developing and delivering crop varieties. In the last two decades, modern biotechnological tools have been developed which led to the better understanding of the genetics of traits and are able to assist conventional breeding to release new cultivars in a shorter span of time. Thus, integrating modern biotechnological tools into the conventional breeding offers new opportunities to breed crop cultivars with enhanced quality, quantity, and tolerance to abiotic and biotic stresses. Besides, locally adapted, several landraces can be developed through plant domestication, farmer’s selection, and self- and cross-pollinated methods. In this chapter, conventional breeding methods, modern and advanced biotechnological tools, viz., plant tissue culture, genetic transformation, hybrids generation, TILLING, RNAi, genome editing, and nano-biotechnology along with their importance will be discussed which would aid in transferring knowledge from scientist to farmer fields.KeywordsPlant breedingPlant tissue cultureMicro-propagationSpeed breedingStress resistance

P03-60 - Levetiracetam showed lower depression, insomnia and mood instability incidence than phenytoin after supratentorial tumour neurosurgery

This study prioritised sustainable agricultural practices in Ebonyi State, Nigeria by using a participatory methodology and selected 240 farmers from the state for interview and group discussion through a multistage sampling technique. Scoring and bidding exercises using pseudo money were employed to check the willingness to pay for various SAPs. Stated preference and chi-square were adopted for data analysis. Strong similarities and differences characterise preferences of different SAPs by farmers in different agricultural zones. Insurance, climate change-based advisory services, improved crop varieties, mounds and adjusting planting dates were the commonly preferred practices. Farmers' scoring and bidding differed significantly for insurance, improved crop varieties and climate change-based advisory services. Adopting a stated preference methodology in prioritising farmers' preferences for sustainable agricultural practices in a changing climate provides relevant information on climate risk management.

Development of a Haploid-Inducer Mediated Genome Editing System for Accelerating Maize Breeding

Oilseed crops are an important source of dietary fats and proteins in humans and animals. They have significant economic importance being the major source of hydrocarbons for the manufacturing of biofuels and industrially relevant bioproducts. Conventional plant breeding methods along with molecular breeding and transgenic technologies has contributed significantly towards the development of high-yielding cultivars of crops, including oilseeds. However, while these methods are cumbersome and time consuming, the genetically modified (GM) crop cultivars are currently not widely accepted due to regulatory concerns. To satisfy the global demand of improved oilseed crops for the ever-growing population, it is essential that alternative approaches to crop improvement must be considered. Plant breeders are now increasingly inclined towards the recently available genome editing tools for the improvement of agriculturally important traits. Among the several gene-editing platforms, the clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system has emerged as a revolutionary genome editing tool for its simplicity and wide acceptability to achieve transgene-free gene modifications. In this review, we focus on understanding the historical development of genome editing tools and molecular mechanism of CRISPR-Cas genome editing system followed by its application for the improvement of various desirable traits in oilseed crops.KeywordsOilseedDietBiofuelsGenetic engineeringGenome editingCRISPR-Cas

A highly tumor-specific light-triggerable drug carrier responds to hypoxic tumor conditions for effective tumor treatment

BackgroundThe genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants.ResultsHere, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1.ConclusionsOur results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.

Genetic divergence, defined as the accumulation of genetic differences between populations, holds fundamental importance in plant breeding and is crucial for the development of new and improved crop varieties. The factors that promote genetic divergence between populations consist of natural selection along with genetic drift together with mutation and gene flow and controlled breeding techniques. The study of genetic divergence employs four distinct analytical approaches which include morphological traits together with cytological markers and biochemical markers and molecular markers as well as genomic data. Studies of finger millet and chickpea along with rice and soybean farming confirm that genetic divergence serves as a key tool for finding distinct genetic elements as well as important traits for conservation. The article surveys the estimation techniques alongside statistical programs which aid genetic divergence determination. Plant breeders can achieve better adapted crop varieties through population diversity conservation which leads to enhanced food security and sustainable agriculture. The detailed content overview gives important findings to both researchers and breeders who want to increase crop variety diversity and adaptiveness against environmental changes.

BackgroundObesity is a worldwide epidemic affecting millions of people. The current pharmacological treatment of obesity remains limited and ineffective due to drugs’ undesirable side effects. Hence, there is a need for novel or improved strategies for long-term therapies that will help prevent the disease progression into other chronic diseases. Nanotechnology holds the future for the treatment of obesity because of its versatility, as shown by improved drug efficiency and safety in cancer clinical trials. Nano-based drug delivery systems could potentially do the same for obesity through targeted drug delivery. This study investigated the use of peptide-functionalized quantum dots (QDs) for the imaging of prohibitin (PHB)-expressing cells in vitro and in diet-induced obese rats, which could potentially be used as nanocarriers of antiobesity drugs.MethodsCadmium (Cd)-based QDs were functionalized with an adipose homing peptide (AHP) and injected intravenously into lean and obese Wistar rats. Biodistribution of the QDs was analyzed by an IVIS® Lumina XR imaging system and inductively coupled plasma optical emission spectroscopy (ICP-OES). For in vitro studies, PHB-expressing (Caco-2 and MCF-7) and non-PHB-expressing (KMST-6 and CHO) cells were exposed to either unfunctionalized QDs (QD625) or AHP-functionalized QDs (AHP-QD625) and analyzed by fluorescence microscopy.ResultsAHP-QD625 accumulated significantly in PHB-expressing cells in vitro when compared with non-PHB-expressing cells. In vivo data indicated that QD625 accumulated mainly in the reticuloendothelial system (RES) organs, while the AHP-QD625 accumulated mostly in the white adipose tissues (WATs).ConclusionAHP-functionalized QDs were successfully and selectively delivered to the PHB-expressing cells in vitro (Caco-2 and MCF-7 cells) and in the WAT vasculature in vivo. This nanotechnology-based approach could potentially be used for dual targeted drug delivery and molecular imaging of adipose tissues in obese patients in real time.

While chimeric antigen receptor (CAR) T cell therapy has shown promising outcomes among patients with hematologic malignancies, it has also been associated with undesirable side-effects such as cytokine release syndrome (CRS). CRS is triggered by CAR T-cell-based activation of monocytes, which are stimulated via the CD40L–CD40R axis or via uptake of GM-CSF to secrete proinflammatory cytokines. Mouse models have been used to model CRS, but working with them is labor-intensive and they are not amenable to screening approaches. To overcome this challenge, we established two simple cell-based CRS in vitro models that entail the co-culturing of leukemic B cells with CD19-targeting CAR T cells and primary monocytes from the same donor. Upon antigen encounter, CAR T cells upregulated CD40L and released GM-CSF which in turn stimulated the monocytes to secrete IL-6. To endorse these models, we demonstrated that neutralizing antibodies or genetic disruption of the CD40L and/or CSF2 loci in CAR T cells using CRISPR-Cas technology significantly reduced IL-6 secretion by bystander monocytes without affecting the cytolytic activity of the engineered lymphocytes in vitro. Overall, our cell-based models were able to recapitulate CRS in vitro, allowing us to validate mitigation strategies based on antibodies or genome editing.

Biotic stresses and abiotic stress factors such as salt, drought, cold and extreme temperatures severely limit crop productivity. Attempts to improve crop varieties for traits conferring tolerance to these stresses are being made continuously to sustain the food production. Molecular breeding and genetic engineering strategies are serving as efficient tools to accelerate the process of developing stress tolerant genotypes. Developing crop varieties that can withstand incidence of multiple stresses is one of the major breeding objectives nowadays, wherein series of target genes identified in different parents are accumulated into a single genotype (gene pyramiding). Generation of transgenic plants by introducing two or three foreign genes is one of the common means to develop plants exhibiting resistance against multiple stresses. Recently there have been advances in the mapping of genes controlling quantitative traits, through quantitative trait loci (QTL) mapping experiments and analysis of genomic data. Such advancements in genome mapping and high-throughput laboratory genotyping protocols have enabled us to monitor introgression of desired genes/regions from two or three different sources into a single background. In this paper, we have attempted to review the achievements made in the field of generation of improved crop varieties by pyramiding (genetic transformation and marker assisted gene pyramiding) desired genes from different sources.

Banana (Musa spp.) is vital global fruit crop that is prone to significant biotic and abiotic stresses. Modern biotechnological tools, such as genome editing and genetic transformation, offer promising strategies to enhance banana resilience. Recent advancements have enabled the transformation of banana plants with desirable traits. Among these methods, Agrobacterium-mediated transformation of embryogenic calli has emerged as the most feasible and cost-effective technique. Optimizing this method for specific cultivars is crucial to improve transformation efficiency. This chapter outlines a successful genetic transformation protocol for the Grand Naine cultivar of banana, a commercially important dessert variety. Emphasizing Agrobacterium-mediated transformation of embryogenic calli, we highlight its economic viability and efficiency.

Citrus is globally one of the major fruit crops, occupying a place of prominence in international trade and tariff through horticultural commodities. Despite such distinction, citrus crop is confronted with a variety of biotic and abiotic stresses, thereby, sustaining production is always a daunting task. The genome size of citrus is rather small, ranging from 265 to 400 MB, probably an advantage for controlled trait specific editing. The evolution of next generation sequencing has facilitated the whole genome sequencing of as many 10 citrus species with 16 draft genome sequences, offering near future possibility to develop genome tailored citrus species or inducing the desired genetic transformation to address the issues chronically ailing commercial citrus cultivation in India, which is by no mean, a simple task to accomplish. Despite genetically intrinsic challenges involved in generating transgenics in perennial crop like citrus, several transgenics have been developed in namely, sweet orange, lemon, and grapefruit loaded with some useful traits. But, the public perception and the time taken to develop trangenics in citrus and less success ratio led the researchers adapt alternate ways. Of late, the thumping success of genome editing tools, especially Clustered. Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has provided a new molecular tailoring machine for citrus improvement. In several citrus species like sweet orange, pummelo, and grapefruit CRISPR-Cas9 system has resulted in value added multiple traits-based transgenics. However, the major drawback of the CRISPR/Cas9 system is the generation of significant off-target cleavage sites as a result of complexing of gRNA with mismatched complementary target DNA within the genome. The use of CRISPR as genome editing technology is anticipated to induce many desired traits in citrus in years to come with more commercial applications in field for changed canopy structure, root traits, regular bearing, extended fruit maturity, besides multiple disease resistance.

In this review, we have focused on the CRISPR/Cas9 technology for improving the agronomic traits in plants through point mutations, knockout, and single base editing, and we highlighted the recent progress in plant metabolic engineering. CRISPR/Cas9 technology has immense power to reproduce plants with desired characters and revolutionizing the field of genome engineering by erasing the barriers in targeted genome editing. Agriculture fields are using this advance genome editing tool to get the desired traits in the crops plants such as increase yield, improve product quality attributes, and enhance resistance against biotic and abiotic stresses by identifying and editing genes of interest. This review focuses on CRISPR/Cas-based gene knockout for trait improvement and single base editing to boost yield, quality, stress tolerance, and disease resistance traits in crops. Use of CRISPR/Cas9 system to facilitate crop domestication and hybrid breeding are also touched. We summarize recent developments and up-gradation of delivery mechanism (nanotechnology and virus particle-based delivery system) and progress in multiplex gene editing. We also shed lights in advances and challenges of engineering the important metabolic pathways that contain a variety of dietary metabolites and phytochemicals. In addition, we endorsed substantial technical hurdles and possible ways to overcome the unpredictability of CRISPR/Cas technology for broader application across various crop species. We speculated that by making a strong interconnection among all genomic fields will give a gigantic bunt of knowledge to develop crop expressing desired traits.