CRISPR-Cas9 Multiplex Editing of the α-Amylase/Trypsin Inhibitor Genes to Reduce Allergen Proteins in Durum Wheat

Abstract

Wheat and its derived foods are widespread, representing one of the main food sources globally. During the last decades, the incidence of disorders related to wheat has become a global issue for the human population, probably linked to the spread of wheat-derived foods. It has been ascertained that structural and metabolic proteins, like α-amylase/trypsin inhibitors (ATI), are involved in the onset of wheat allergies (bakers' asthma) and probably Non-Coeliac Wheat Sensitivity (NCWS). The ATI are a group of exogenous protease inhibitors, which are encoded by a multigene family dispersed over several chromosomes in durum and bread wheat. WTAI-CM3 and WTAI-CM16 subunits are considered among the main proteins involved in the onset of bakers' asthma and probably NCWS. A CRISPR-Cas9 multiplexing strategy was used to edit the ATI subunits WTAI-CM3 and WTAI-CM16 in the grain of the Italian durum wheat cultivar Svevo with the aim to produce wheat lines with reduced amount of potential allergens involved in adverse reactions. Using a marker gene-free approach, whereby plants are regenerated without selection agents, homozygous mutant plants without the presence of CRISPR vectors were obtained directly from T 0 generation. This study demonstrates the capability of CRISPR technology to knock out immunogenic proteins in a reduced time compared to conventional breeding programmes. The editing of the two target genes was confirmed either at molecular (sequencing and gene expression study) or biochemical (immunologic test) level. Noteworthy, as a pleiotropic effect, is the activation of the ATI 0.28 pseudogene in the edited lines.

The α-Gliadins in Bread Wheat: Effect of Nitrogen Treatment on the Expression of the Major Celiac Disease Immunogenic Complex in Two RNAi Low-Gliadin Lines

“…The application of biotechnological techniques such as RNAi and CRISPR/Cas provided new wheat varieties with immunogenic gliadins strongly down-regulated ( Barro et al, 2016 ; Sánchez-León et al, 2018 ) showing that these techniques are appealing alternatives for the development of gluten-free cereal varieties that can be tolerated by celiacs or patients suffering other gluten-related pathologies ( Haro et al, 2018 ; Camerlengo et al, 2020 ). For RNAi varieties, the decrease in the content of α-gliadins was up to 90% ( Pistón et al, 2013 ).…”

Quantitative Label-Free Comparison of the Metabolic Protein Fraction in Old and Modern Italian Wheat Genotypes by a Shotgun Approach

“…Moreover, the genotype Timilia presents a much higher relative abundance of αamylase inhibitor 0.28 in comparison with the other two genotypes investigated here. This finding appears very interesting because this protein represents one of the non-gluten components, listed in the Allergome database (http://www.allergome.org/, accessed on date 8 February 2021), likely to be allergenic, and may have a role in nonceliac wheat sensitivity (NCWS) and celiac disease (CD) [54][55][56].…”

Reduction of Allergenic Potential in Bread Wheat RNAi Transgenic Lines Silenced for CM3, CM16 and 0.28 ATI Genes

“…Recently we have obtained durum wheat lines in which the ATI genes CM3 and CM16 have been silenced by CRISPR-Cas9 multiple editing [16], and that are now under multiplication and characterization. Differently, in the present work, we report the characterization, in terms of protein and gene expression, of allergenic potential, and of parameters related to technological performances of bread wheat lines in which three genes coding for ATI proteins, namely CM3, CM16 and 0.28, involved in BA and likely in NCWS, have been silenced by RNAi.…”

“…The availability of genotypes with reduced amounts of ATI proteins might allow either the development of novel wheat cultivars with a lower triggering potential, or a better understanding of the role of these components in such wheat-related pathologies. Recently, our group [16] has reported the application of CRISPR-Cas9 silencing technology to silence CM3 and CM16 ATI genes in durum wheat, whereas in this paper we report the development and the extensive characterization, including the allergenic potential and qualitative evaluations, of wheat lines derived from the bread wheat cultivar Bobwhite, in which CM3, CM16 and 0.28 ATI genes have been silenced by RNAi.…”