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Celiac.com 10/21/2024 - Wheat has been a staple in human diets for centuries, but for individuals with celiac disease and gluten sensitivities, consuming wheat can lead to serious health complications. The culprit? Gluten, particularly the gliadin fractions found in wheat, which trigger immune responses in those affected by celiac disease. A new study presents groundbreaking advancements in the field of gluten-free wheat production. Researchers have successfully used CRISPR/Cas9 technology to target and reduce gluten content by editing the genes responsible for the immunogenic gliadin proteins. Targeting Gliadin Genes with CRISPR/Cas9 The researchers in this study focused on two major groups of gliadin proteins: gamma (γ) and omega (ω) gliadins. These proteins are known to contain key epitopes that trigger adverse immune reactions in people with celiac disease. By designing eight specific single guide RNAs (sgRNAs), they were able to target and introduce mutations into these gliadin genes. Through the use of CRISPR/Cas9 multiplexing, a total of 59 wheat lines were produced, with 20 showing successful mutations in the target genes. This step marked a significant advance in reducing the gluten content of wheat. Massive Reduction in Gluten Content One of the most striking findings of the study was the 97.7% reduction in gluten content in the edited wheat lines. This was confirmed through various methods, including Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) and monoclonal antibodies. The reduction was most noticeable in γ-gliadins and ω1,2-gliadins, while α-gliadins saw smaller decreases. These reductions make the modified wheat an incredibly promising candidate for gluten-free food products, although some limitations remain when it comes to fully eliminating all immunogenic components. Combining Multiple Mutations In addition to targeting γ- and ω-gliadins, the researchers took an extra step by crossing these modified lines with CRISPR/Cas9 lines that had already been edited for α-gliadins. This approach resulted in wheat lines with multiple mutations across different gliadin families, further enhancing the reduction of gluten content. By combining multiple edits, the research has moved closer to producing wheat that is significantly safer for people with celiac disease or gluten sensitivities. Quantifying the Success: R5 and G12 Monoclonal Antibody Tests To measure the gluten content in the edited wheat lines, the study used two monoclonal antibody tests, R5 and G12, which are standard in the food industry for detecting gluten in products. The R5 antibody is specifically raised against rye ω-secalins, which are similar to wheat ω-gliadins, while the G12 antibody targets α-gliadins. Both tests showed significant reductions in gluten, with some lines showing up to 97.7% less gluten. However, there were discrepancies between the two tests, with the G12 test showing slightly higher gluten content. This suggests that while the wheat lines are substantially gluten-reduced, there is still room for further refinement in achieving a completely gluten-free product. Future Implications: The Path to Non-Transgenic, Immune-Safe Wheat While previous efforts had been made to develop low-gluten wheat, this study is notable for demonstrating the feasibility of using CRISPR/Cas9 to edit multiple genes simultaneously in polyploid bread wheat. The result is a collection of wheat lines with drastically reduced levels of immunogenic proteins, moving closer to the goal of producing wheat that is safe for individuals with celiac disease. Importantly, these modified wheat lines are non-transgenic, meaning they do not contain foreign DNA, which may make them more acceptable to consumers and regulators. The next steps involve further testing, including stimulation assays using peripheral blood mononuclear cells (PBMCs) from individuals with celiac disease. These tests will help researchers identify which wheat lines cause the least immune response, with the ultimate goal of producing wheat that can be safely used in gluten-free products. A Step Toward Gluten-Free Wheat for Celiac Disease Patients For individuals with celiac disease, the promise of gluten-free wheat represents a potential game-changer. This study has made significant strides toward that goal by reducing the immunogenic gliadin proteins that cause adverse reactions. While further testing and development are necessary, the application of CRISPR/Cas9 technology in wheat breeding holds great promise for the future of gluten-free diets. The study does not specifically address the baking properties of the new wheat varieties with reduced gluten content. However, this is an essential consideration when developing gluten-free or gluten-reduced wheat. Gluten plays a critical role in baking, as it provides dough with its elasticity, structure, and ability to trap air during fermentation, leading to the light and airy texture of bread and other baked goods. When gluten is removed or reduced, it can significantly alter these properties, resulting in denser, less cohesive baked products. Therefore, it's crucial that any new gluten-free wheat varieties maintain similar baking characteristics, or alternative methods, such as adding binders like xanthan gum or psyllium husk, may be needed to replicate the qualities gluten imparts in baking. Balancing the reduction of immunogenic proteins with maintaining functional qualities is key to making the wheat practical for food production. This research is especially meaningful for those with celiac disease because it offers hope for a future where they can enjoy wheat-based foods without fear of triggering harmful immune responses. By creating wheat with drastically reduced gluten content, researchers are opening doors to new gluten-free food options that could improve the quality of life for millions of people worldwide. Read more at: academic.oup.com

Celiac.com 01/08/2018 - Imagine gluten-free wheat. Well, actually you don't have to imagine it, because a group of scientists has used a gene-editing technique called CRISPR/Cas9 to cut selected genes from a wheat genome, and presto, gluten-free wheat is a thing. As people in numerous countries debate genetically modified crops, some countries, including France and Germany, have passed laws to prohibit their cultivation. Remember, we're not talking about hybridization here, which is based on natural selection and works by interbreeding plant strains. Researchers have used hybridization to develop strains of wheat that are low in gluten, but so far no one has made a strain that is entirely free of gluten. In this case, we're talking about genetic modification; changing the basic genetic structure of the plant. The greatest objections around GMO practices have been focused on the insertion of DNA from one species into another species, says Francisco Barro, a plant biotechnologist at the Institute for Sustainable Agriculture in Spain. To steer clear of this genetic process, Barro and his team used the gene-editing technique CRISPR/Cas9 to remove certain genes from a wheat genome. Their team focused on alpha-gliadins, gluten proteins that are thought to be the trigger for immune system reactions in people with celiac disease and gluten sensitivity. To accomplish their goal of removing the culprit gene(s), the research team used the scissorlike Cas9 protein to cut out 35 of the 45 alpha-gliadin genes. Lab tests showed that the new wheat strain reduced the immune response by 85 percent, the team reported. Far from being any kind of decisive breakthrough though, this is just one “really important step in maybe producing something that is going to be incredibly useful,” says Wendy Harwood, a crop geneticist at the John Innes Center in England. Meanwhile, Barro says his team is working on targeting more gluten-triggered genes to develop a completely safe strain of wheat for celiac patients. Source: Scientific American

Celiac.com 10/25/2017 - For people with celiac disease, eating gluten proteins from wheat, barley, and rye triggers an auto-immune response, and the accompanying physical symptoms. A team of researchers recently set out to engineer low-gluten wheat strains that also have low-reactivity for people with celiac disease. To meet their goals, the team designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33-mer in the -gliadin genes. They then sought to evaluate the results. The research team included Susana Sánchez-León, Javier Gil-Humanes, Carmen V. Ozuna, María J. Giménez, Carolina Sousa, Daniel F. Voytas, and Francisco Barro. They are variously affiliated with the Departamento de Mejora Genética Vegetal, Instituto de Agricultura Sostenible (IAS-CSIC), Córdoba, Spain; the Department of Genetics, Cell Biology, and Development, Center for Genome Engineering at the University of Minnesota in Minneapolis, MN, USA; and with the Facultad de Farmacia, Departamento de Microbiología y Parasitología, Universidad de Sevilla, Spain. The gliadin gene family of wheat includes four highly reactive peptides, with the 33-mer peptide being the main culprit in celiac patients. In all, the team generated twenty-one mutant lines, all of which showed strong reduction gliadin proteins. Of the 45 different genes identified in wild type gliadin, the team mutated up to 35 different genes in one of the lines to achieve an 85% reduction in immunoreactivity. They then identified the transgene-free lines, and found no off-target mutations in any of the potential targets. So, what does this all mean in English? Well, basically the low-gluten, transgene-free wheat lines that the team describes here could be used to produce low-gluten foods, as well as serving as source material to introduce the low-gluten, low-reactivity traits into selected wheat varieties. Basically, the technology could be used to create low-gluten wheat varieties with low immunoreactivity. Now, most folks with celiac disease, especially those with higher gluten sensitivity, would likely need more than and 85% reduction in immunoreactivity to see any real benefit. However, this study provides an interesting glimpse at how science might help researchers to create wheat strains that are safe for people with celiac disease. Source: Plant Biotechnology Journal. DOI: 10.1111/pbi.12837