Field assessment of genome‐edited, low asparagine wheat: Europe's first <scp>CRISPR</scp> wheat field trial

Raffan, Sarah; Oddy, Joseph; Meade, A.; Barker, G. L.; Curtis, Tanya Y.; Usher, Sarah; Burt, Christopher; Halford, Nigel G.

doi:10.1111/pbi.14026

Cited by 17 publications

(8 citation statements)

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“…Evolutionary selection favours mutations in genes with low pleiotropy, that are expressed in a small number of tissues, and which are predicted to be associated with few biological processes ( Stern and Orgogozo, 2008 ). Reported applications of CRISPR/Cas9 in wheat including the PPO1 and PPO2 genes described here, as well as TaASN2 ( Raffan et al., 2023 ) and glutenin genes ( Yu et al., 2023 ) match this profile. An underexplored source of adaptive mutations are gain-of-function alleles that affect transcriptional regulation ( Martin and Orgogozo, 2013 ) as demonstrated previously ( Rodríguez-Leal et al., 2017 ; Song et al., 2022 ).…”

Section: Discussionmentioning

confidence: 64%

Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivum L.) grains

Wold-McGimsey,

Krosch,

Alarcón-Reverte

et al. 2023

Front. Plant Sci.

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IntroductionPolyphenol oxidases (PPO) are dual activity metalloenzymes that catalyse the production of quinones. In plants, PPO activity may contribute to biotic stress resistance and secondary metabolism but is undesirable for food producers because it causes the discolouration and changes in flavour profiles of products during post-harvest processing. In wheat (Triticum aestivum L.), PPO released from the aleurone layer of the grain during milling results in the discolouration of flour, dough, and end-use products, reducing their value. Loss-of-function mutations in the PPO1 and PPO2 paralogous genes on homoeologous group 2 chromosomes confer reduced PPO activity in the wheat grain. However, limited natural variation and the proximity of these genes complicates the selection of extremely low-PPO wheat varieties by recombination. The goal of the current study was to edit all copies of PPO1 and PPO2 to drive extreme reductions in PPO grain activity in elite wheat varieties.ResultsA CRISPR/Cas9 construct with one single guide RNA (sgRNA) targeting a conserved copper binding domain was used to edit all seven PPO1 and PPO2 genes in the spring wheat cultivar ‘Fielder’. Five of the seven edited T1 lines exhibited significant reductions in PPO activity, and T2 lines had PPO activity up to 86.7% lower than wild-type. The same construct was transformed into the elite winter wheat cultivars ‘Guardian’ and ‘Steamboat’, which have five PPO1 and PPO2 genes. In these varieties PPO activity was reduced by >90% in both T1 and T2 lines. In all three varieties, dough samples from edited lines exhibited reduced browning.DiscussionThis study demonstrates that multi-target editing at late stages of variety development could complement selection for beneficial alleles in crop breeding programs by inducing novel variation in loci inaccessible to recombination.

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Section: Discussionmentioning

confidence: 64%

Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivum L.) grains

Wold-McGimsey,

Krosch,

Alarcón-Reverte

et al. 2023

Front. Plant Sci.

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“…Evolutionary selection favours mutations in genes with low pleiotropy, that are expressed in a small number of tissues, and which are predicted to be associated with few biological processes (Stern and Orgogozo, 2008). Reported applications of CRISPR/Cas9 in wheat including the PPO1 and PPO2 genes described here, as well as TaASN2 (Raffan et al, 2023) and glutenin genes (Yu et al, 2023) match this profile. An underexplored source of adaptive mutations are gain-of-function alleles that affect transcriptional regulation (Martin and Orgogozo, 2013) as demonstrated previously (Rodríguez-Leal et al, 2017;Song et al, 2022).…”

Section: Applications In Breedingmentioning

confidence: 58%

Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivumL.) grains

Wold-McGimsey,

Krosch,

Reverte

et al. 2023

Preprint

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Polyphenol oxidases (PPO) are dual activity metalloenzymes that catalyse the production of quinones. In plants, PPO activity may contribute to biotic stress resistance and secondary metabolism but is undesirable for food producers because it causes the discolouration and changes in flavour profiles of products during post-harvest processing. In wheat (Triticum aestivum L.), PPO released from the aleurone layer of the grain during milling results in the discolouration of flour, dough, and end-use products, reducing their value. Loss-of-function mutations in the PPO1 and PPO2 paralogous genes on homoeologous group 2 chromosomes confer reduced PPO activity in the wheat grain but limited natural variation and small intergenic distances between these genes complicates the selection of extremely low-PPO wheat varieties by recombination. In the current study, a CRISPR/Cas9 construct with one single guide RNA (sgRNA) targeting a conserved copper binding domain was used to edit all seven PPO1 and PPO2 genes in the spring wheat cultivar Fielder. Five of the seven edited T1 lines exhibited significant reductions in PPO activity, and T2 lines had PPO activity up to 86.7% lower than wild-type controls. In the elite winter wheat cultivars Guardian and Steamboat, which have five PPO1 and PPO2 genes, PPO activity was reduced by >90% in both T1 and T2 lines. This study demonstrates that multi-target editing at late stages of variety development could complement selection for beneficial alleles in crop breeding programmes by inducing novel variation in loci inaccessible to recombination.

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“…The mean free asparagine concentrations in the total TaASN2 nulls were just under 50% that of the Cadenza control, while in the A genome nulls the concentration was 86% that of Cadenza [67]. The TILLING lines showed more variable responses: they did show a reduction of 20-40% compared with a TILLING control (a line that had come through the same process but did not carry mutations in TaASN2), but Claire was also lower than the TILLING control, so further trials will be required before firm conclusions can be drawn.…”

Section: The Application Of Genome Editing and Chemical Mutagenesis T...mentioning

confidence: 83%

“…The editing was achieved by introducing a 4-gRNA polycistronic gene into wheat embryos along with a Cas9 gene and a marker gene (Bar). Free asparagine concentration in plants in which all six TaASN2 alleles were knocked out was 43-57% of the wildtype over two generations [66], and in plants that were subsequently found to be edited in TaASN1 as well as TaASN2 (i.e., TaASN1/TaASN2 total knockouts) [67] was 9-48% of the wildtype [65]. In plants containing edits only in the A genome TaASN2 alleles, the concentration of free asparagine was 56-68% of wildtype [66].…”

Section: The Application Of Genome Editing and Chemical Mutagenesis T...mentioning

confidence: 99%

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Reducing the Risk of Acrylamide and Other Processing Contaminant Formation in Wheat Products

Kaur,

Halford

2023

Foods

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Wheat is a staple crop, consumed worldwide as a major source of starch and protein. Global intake of wheat has increased in recent years, and overall, wheat is considered to be a healthy food, particularly when products are made from whole grains. However, wheat is almost invariably processed before it is consumed, usually via baking and/or toasting, and this can lead to the formation of toxic processing contaminants, including acrylamide, 5-hydroxymethylfurfural (HMF) and polycyclic aromatic hydrocarbons (PAHs). Acrylamide is principally formed from free (soluble, non-protein) asparagine and reducing sugars (glucose, fructose and maltose) within the Maillard reaction and is classified as a Group 2A carcinogen (probably carcinogenic to humans). It also has neurotoxic and developmental effects at high doses. HMF is also generated within the Maillard reaction but can also be formed via the dehydration of fructose or caramelisation. It is frequently found in bread, biscuits, cookies, and cakes. Its molecular structure points to genotoxicity and carcinogenic risks. PAHs are a large class of chemical compounds, many of which are genotoxic, mutagenic, teratogenic and carcinogenic. They are mostly formed during frying, baking and grilling due to incomplete combustion of organic matter. Production of these processing contaminants can be reduced with changes in recipe and processing parameters, along with effective quality control measures. However, in the case of acrylamide and HMF, their formation is also highly dependent on the concentrations of precursors in the grain. Here, we review the synthesis of these contaminants, factors impacting their production and the mitigation measures that can be taken to reduce their formation in wheat products, focusing on the role of genetics and agronomy. We also review the risk management measures adopted by food safety authorities around the world.

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Field assessment of genome‐edited, low asparagine wheat: Europe's first CRISPR wheat field trial

Cited by 17 publications

References 5 publications

Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivum L.) grains

Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivum L.) grains

Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivumL.) grains

Reducing the Risk of Acrylamide and Other Processing Contaminant Formation in Wheat Products

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