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| Applications of CRISPR editing in allergy research. To date, CRISPR technology has been applied to edit allergen genes in cat, hen's egg, soybean, wheat, peanut, and cow's and goat's milk (ATIs: α-amylase/trypsin inhibitors).
Source publication
Genome engineering with clustered regularly interspaced short palindromic repeats (CRISPR) technology offers the unique potential for unequivocally deleting allergen genes at the source. Compared to prior gene editing approaches, CRISPR boasts substantial improvements in editing efficiency, throughput, and precision. CRISPR has demonstrated success...
Context in source publication
Context 1
... editing has shown promise in numerous applications of allergy research (Figure 2). These studies demonstrate the value of the technology in improving our understanding of allergen proteins, and underscore the vast potential for CRISPR editing to provide better, alternative treatment options for allergic disease. ...
Citations
... Gene editing is a potent instrument, especially advantageous when aimed at deleterious genes that present health hazards to organisms like humans, animals, or plants and their progeny [48]. In agriculture, gene editing facilitates the creation of crops that resist pests and diseases, thereby bolstering food security and diminishing dependency on chemical pesticides [49]. Additionally, gene editing shows potential in tackling food allergens, potentially eradicating risks linked to allergic responses [50]. ...
... The existence of so-called hypoallergenic animals and their suitability as pets or companion animals are often a matter of great interest and discussion. 14,[32][33][34][35] The present study aimed to investigate differences in protein and allergen content of hair extracts from the Curly Horse, Our data are in line with previous studies on so-called hypoallergenic animals. Until now, there is no scientific evidence for the existence of hypoallergenic cat, dog, and horse breeds. ...
Background
The American Bashkir Curly Horse is frequently advertised to horse‐allergic riders and claimed to be a so‐called hypoallergenic breed that elicits fewer symptoms. Previous studies quantifying selected allergens in different breeds did not find a reduced allergen content in Curly Horses. Here, we provide a comprehensive proteomic analysis of horse hair extracts and a molecular analysis of the major allergen Equ c 1 with the aim of identifying differences in the Curly Horse breed that might explain their presumed reduced allergenic potential.
Methods
Horse hair extracts were prepared from Curly and American Quarter Horse breeds, separated by gender and castration status, extracts from other breeds served as controls. Extracts and native Equ c 1 (nEqu c 1) were analyzed by mass spectrometry. IgE‐binding capacities of nEqu c 1 and its recombinant variants were tested by ELISA using sera of patients sensitized to horses. Structures and ligand binding abilities were analyzed by computational modeling and fluorescence quenching assays.
Results
All known respiratory horse allergens are present in hair extracts of Curly and Quarter Horses and share identical allergen‐specific peptides. Lipocalin allergens are the most abundant proteins in horse hair extracts and contain several post‐translational modifications. We identified two new variants of Equ c 1 that have similar IgE‐binding capacities but show structural differences in their binding cavities and altered ligand binding behavior. There are no differences in IgE‐binding of Equ c 1 derived from Curly Horses compared to other horse breeds.
Conclusion
Our data do not support the claim that Curly Horses are less allergenic than other breeds.
... 31 Promising steps toward gene editing of allergen sequences using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology have been reported. 41 This review focuses on current strategies for effectively identifying allergenic epitopes, for localization and definition of contact residues, and for validation of the biologic importance of IgE epitope(s) by genetic engineering and geneediting techniques. ...
... 108 To date, CRISPR has been applied for the targeted deletion of allergens with the aim of better understanding allergen proteins and providing improved, alternative treatment options for allergic disease. 41 Several recent applications have demonstrated the feasibility of knocking out inhaled allergens using CRISPR-Cas9, such as the development of pollen-free Japanese cedar trees. 109 CRISPR-Cas9 was used to knock out the major allergen produced by domestic cats, Fel d 1, in a nonexpressing cell line. ...
... In addition to RNAi, the use of technological advancement, the discovery and characterization of plant food allergen genes offers a significant opportunity for successful genetic modifications (Brackett et al., 2022). Biotechnological techniques, such as gene editing or genome editing, are widely employed to produce designer crops with desired traits Fernie and Yan, 2019;Awasthi et al., 2022). ...
In addition to the challenge of meeting global demand for food production, there are increasing concerns about food safety and the need to protect consumer health from the negative effects of foodborne allergies. Certain bio‐molecules (usually proteins) present in food can act as allergens that trigger unusual immunological reactions, with potentially life‐threatening consequences. The relentless working lifestyles of the modern era often incorporate poor eating habits that include readymade prepackaged and processed foods, which contain additives such as peanuts, tree nuts, wheat, and soy‐based products, rather than traditional home cooking. Of the predominant allergenic foods (soybean, wheat, fish, peanut, shellfish, tree nuts, eggs, and milk), peanuts (Arachis hypogaea) are the best characterized source of allergens, followed by tree nuts (Juglans regia, Prunus amygdalus, Corylus avellana, Carya illinoinensis, Anacardium occidentale, Pistacia vera, Bertholletia excels), wheat (Triticum aestivum), soybeans (Glycine max), and kidney beans (Phaseolus vulgaris). The prevalence of food allergies has risen significantly in recent years including chance of accidental exposure to such foods. In contrast, the standards of detection, diagnosis, and cure have not kept pace and unfortunately are often suboptimal. In this review, we mainly focus on the prevalence of allergies associated with peanut, tree nuts, wheat, soybean, and kidney bean, highlighting their physiological properties and functions as well as considering research directions for tailoring allergen gene expression. In particular, we discuss how recent advances in molecular breeding, genetic engineering, and genome editing can be used to develop potential low allergen food crops that protect consumer health.
... Cas9 has commonly been used in studies to evaluate allergens and immune-related diseases such as egg allergies [148], or to remove allergens from products such as soybean through site-directed mutagenesis [149] or target genes involved in allergic rhinitis [150]; or remove and functionally evaluate other allergens [151][152][153][154][155][156], with no literature studies having implicated Cas9 proteins themselves in allergenicity. Importantly, Nakajima et al. [54] reported that Cas9 was subject to rapid digestion in vitro and unlikely to cause food allergy in the case of accidental ingestion. ...
Background
Population suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxFCRISPRh transgene encoding (i) hCas9 endonuclease, (ii) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii) DsRed fluorescent marker protein, in genetically-modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxFCRISPRh previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity.
Methods
Amino acid sequences of hCas9 and DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or DsRed, or of the donor organisms from which these products were originally derived.
Results
While Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence from previous studies of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxFCRISPRh transgenic proteins from environmental releases was assessed as negligible.
Conclusions
Bioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxFCRISPRh were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9.
... Food allergies affect millions of people worldwide and can cause severe and sometimes life-threatening reactions. Gene editing offers a promising solution to this problem by allowing the targeted removal of allergenic proteins from crops without affecting their nutritional value [99,100]. Researchers have used CRISPR/Casbased gene editing to create hypoallergenic peanuts, wheat, and soybeans, among other crops [99,[101][102][103]. ...
... Gene editing offers a promising solution to this problem by allowing the targeted removal of allergenic proteins from crops without affecting their nutritional value [99,100]. Researchers have used CRISPR/Casbased gene editing to create hypoallergenic peanuts, wheat, and soybeans, among other crops [99,[101][102][103]. ...
Food insecurity has become a pressing issue on a worldwide scale as the globe plows through a food crisis. The disastrous impact of this menace has been exacerbated by climate change, frequent conflicts, pandemic outbreaks, and the global economic recession, which have been prevalent in recent years. Although food insecurity prevails globally, it is especially critical in some regions in Africa, East and Southeast Asia, and South America. Several efforts have been made to curb food insecurity; however, none have been able to curtail it sufficiently. Genetic engineering of crops is a fast-growing technology that could be a viable tool for mitigating food insecurity. Crop varieties resistant to pests and diseases, abiotic stress, spoilage, or specific herbicides have been developed using this technology. Crops have been modified for increased yield, nutritional content, essential vitamins, and micro-mineral fortification. More intriguing is the advent of plant-derived edible vaccines, which prove equally effective and significantly affordable. However, in many countries, government policies pose a limiting factor for the acceptance of this technology. This article discusses the genetic modification of crops, highlighting its origins, methods, applications, achievements, impact, acceptance, distribution, and potential as a viable antidote to global food insecurity.
... Food allergies affect between four and 8% of the total population, with over 160 foods known to cause allergic reactions. Allergens are macromolecules, often proteinaceous in nature, that invoke immune responses ranging from mild discomfort (rashes, swelling, gastrointestinal symptoms) to more severe reactions, including swelling of the airways or even fatal anaphylactic shock (Ballmer-Weber, 2011;Brackett et al., 2021). Nine food groups, including milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, wheat, soybeans and sesame, are associated with over 90% of allergic reactions, resulting in statutory requirements for labeling and inspection aimed at reducing their harmful impacts on susceptible consumers. ...
... Of the 32 proteins present in peanuts, 18 of these produce some allergic response, with Ara h 1, Ara h 2, Ara h 3 and Ara h 6 considered to be major allergens, as they can lead to anaphylaxis (Pandey et al., 2019). The glycoprotein Ara h 2, which is recognized by IgE antibodies in 90% of those with peanut allergies, can trigger allergic responses at low concentrations (Brackett et al., 2021). Plasmid-borne RNA interference (RNAi, a "gene silencing" approach) introduced to peanut explants using an Agrobacterium vector, has been used to knock down Ara h 2 mRNA by 25% compared to wild type levels. ...
... Plasmid-borne RNA interference (RNAi, a "gene silencing" approach) introduced to peanut explants using an Agrobacterium vector, has been used to knock down Ara h 2 mRNA by 25% compared to wild type levels. Ongoing work is aimed at removing Ara h 1, 2, 3 and 6 antigens through CRISPR-based editing (Brackett et al., 2021). Critics argue that creation of a completely allergen-free peanut may not be possible or practical, as removing multiple proteins may not only be technically challenging but could also negatively impact the health of the plant and/or nutritional content. ...
... Literature assessment of hCas9 allergenicity Cas9 has commonly been used in studies to evaluate allergens and immune-related diseases such as egg allergies [147], or to remove allergens from products such as soybean through site-directed mutagenesis [148] or target genes involved in allergic rhinitis [149]; or remove and functionally evaluate other allergens [150][151][152][153][154][155], with no literature studies having implicated Cas9 proteins themselves in allergenicity. Importantly, Nakajima et al. [156] reported that Cas9 was subject to rapid digestion in vitro and unlikely to cause food allergy in the case of accidental ingestion. ...
Background
Population suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxFCRISPRh transgene encoding (i) hCas9 endonuclease, (i) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii) DsRed fluorescent marker protein, in genetically modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxFCRISPRh previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity.
Methods
Amino acid sequences of hCas9 and DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or DsRed, or of the donor organisms from which these products were originally derived.
Results
While Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxFCRISPRh transgenic proteins from environmental releases was assessed as negligible.
Conclusions
Bioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxFCRISPRh were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9.
Genome editing, a powerful technology for precise manipulation of DNA sequences, has revolutionized the field of agricultural biotechnology. In recent years, there has been increasing interest in applying genome editing techniques to improve important crop plants, such as peanut (Arachis hypogaea). Peanuts are a vital source of oil and protein, and they play a crucial role in global food security. However, peanut crops face numerous challenges, including susceptibility to diseases, pests, and environmental stressors. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing tools has provided researchers with a rapid, efficient, and precise method to edit the peanut genome. Despite being a polyploid crop, several successful applications of genome editing in peanuts have been reported. For instance, CRISPR/Cas9-mediated gene editing has been used to increase oleic acid content in oil and allergen reduction in peanut varieties through precise genome modifications. However, despite these advancements, challenges remain in the widespread adoption of genome editing in peanuts, off-target effects, and unintended consequences. Advancements in CRISPR-based genome editing holds great promise for the improvement of peanuts by addressing its various prospective traits like phytate reduction, fresh seed dormancy, aflatoxin resistance and abiotic stress tolerance. However, careful consideration of issues, such as safety assessment, and public acceptance, is essential for the successful application and commercialization of genome-edited peanut varieties. Future research and collaborations are needed to overcome these challenges and fully harness the potential of genome editing in improving peanut crops for sustainable agriculture and global food security.
