Miriam Marín-Sanz

The growing prevalence of gluten-related disorders in humans has driven the development of wheat varieties with reduced immunogenic gluten. This study aimed to integrate RNA interference (RNAi) and CRISPR genome editing within a doubled haploid (DH) platform to overcome challenges of gene redundancy and polyploidy in wheat gliadins. We generated DH lines from crosses between RNAi and CRISPR lines and elite wheat cultivars, enabling stable fixation of multiple genetic modifications in a single generation. Deep sequencing analysis of α-gliadin amplicons was conducted using a custom bioinformatics pipeline optimized for complex, repetitive gene families. Gluten protein profiles were evaluated using RP-HPLC and R5 monoclonal antibody. Several DH lines presented >70% reduction in immunogenic epitopes in α-gliadins, with lines outperforming both parents. Editing frequency was influenced by sgRNA efficiency and parental background. Silencing and editing combined led to nearly depleted gliadins in some lines, often with compensatory increases in other storage proteins linked to bread-making quality, such as high-molecular-weight glutenin subunits. Kernel and specific weight traits were largely maintained. This work demonstrates that combining RNAi and CRISPR in a DH platform enables efficient, heritable reduction of immunogenic gluten, providing a viable strategy for breeding wheat lines safer for individuals with gluten-related disorders.

Infections caused by KPC-producing Klebsiella pneumoniae (KPC-KP) represent a persistent public health challenge. This prospective study examines ten-year trends, clinical features, and genomic epidemiology of hospital-onset (HOI) and non-hospital-onset (non-HOI, including healthcare-associated [HcAI] and community-acquired [CA]) KPC-KP infections following a 2012 outbreak. We evaluated the impacts of a 2014 antimicrobial stewardship program (ASP) and COVID-19-related infection prevention and control (IPC) measures, with emphasis on hospital-to-community dissemination. We analysed a prospective, longitudinal cohort of patients (2012–2022) in a tertiary referral hospital. Interrupted time series and ARIMA models assessed ASP and IPC impacts on incidence density (ID). Cross-correlation analysis explored temporal associations between HOI and non-HOI trends. Whole-genome sequencing and PERMANOVA evaluated the genomic structure of ST512/KPC-3 isolates. Multivariable regression analysed the association between infection type and clinical outcomes. Among 467 patients, 33.2% had non-HOI (ID 0.53/1,000 admissions/month) and 66.8% HOI (ID 0.30, p = 0.39). Urinary tract infections predominated in non-HOI (52.9%), while bloodstream and respiratory infections were more common in HOI. Incidence density of HOI and non-HOI infections declined significantly following ASP implementation, with a 4-month lag suggesting sequential transmission dynamics. These reductions were maintained during the pandemic. Genomic data confirmed ST512/KPC-3 dominance and hospital-to-community spread, with temporal factors—rather than acquisition type—explaining genetic variation. Adjusted analyses showed similar 30-day mortality and treatment responses across HOI and non-HOI. ASP and COVID-19 IPC measures contributed to maintaining low KPC-KP incidence. Genomic evidence underscores the role of temporal dynamics and clonal expansion in ST512/KPC-3 dissemination. Non-HOI infections are clinically significant and require targeted, system-wide surveillance and control strategies.

Rice seed storage proteins (SSPs) play a critical role in determining the nutritional quality, cooking properties, and digestibility of rice. To enhance seed quality, CRISPR/Cas9 genome editing was applied to modify SSP composition by targeting genes encoding 13 kDa prolamins and type A glutelins. Three CRISPR/Cas9 constructs were designed: one specific to the 13 kDa prolamin subfamily and two targeting conserved GluA glutelin regions. Edited T₀ and T₁ lines were generated and analyzed using InDel analysis, SDS-PAGE, Bradford assay, and RP-HPLC. Insertions were more frequent than deletions, accounting for 56% and 74% of mutations in prolamin and glutelin genes, respectively. Editing efficiency varied between sgRNAs. All lines with altered protein profiles contained InDels in target genes. SDS-PAGE confirmed the absence or reduction in bands corresponding to 13 kDa prolamins or GluA subunits, showing consistent profiles among lines carrying the same construct. Quantification revealed significant shifts in SSP composition, including increased albumin and globulin content. Prolamin-deficient lines showed reduced prolamins, while GluA-deficient lines exhibited increased prolamins. Total protein content was significantly elevated in all edited lines, suggesting enrichment in lysine-rich fractions. These findings demonstrate that CRISPR/Cas9-mediated editing of SSP genes can effectively reconfigure the rice protein profile and enhance its nutritional value.

Summary: Celiac disease (CD) is a chronic enteropathy affecting approximately 1% of the global population. Wheat α‐gliadins are a major contributor to the autoimmune response, as they contain one of the most immunogenic peptides, the 33‐mer, along with numerous variants. In this study, we used CRISPR/Cas technology to mutate genes encoding α‐gliadins. This approach employed paired sgRNAs to precisely excise immunogenic regions while preserving non‐immunogenic sequences within the α‐gliadins. Furthermore, we replaced the excised region with an α‐gliadin‐based double‐stranded oligodeoxynucleotide (dsODN) designed with nucleotide changes to reduce immunoreactivity and increase peptidase cleavage sites. Two different CRISPR systems, Cas9 and Cas12a, were applied to generate wheat protoplasts and plants with non‐immunogenic regions. Cas9 demonstrated superior performance in terms of editing frequency, excision and replacement of immunogenic fragments. However, the Cas12a nuclease (Cpf1) showed promising editing efficiency, offering the potential for future wheat editing applications. Using the Cas9 system, we achieved a 74.2% excision rate of the 33‐mer in wheat plants. Subsequent analyses showed a significant reduction in the reactivity to the G12 monoclonal antibody, capable of identifying the 33‐mer peptide and a decrease in the prolamin levels compared to the wild‐type. Additionally, we developed a high‐throughput sequencing‐based software specifically designed to identify mutations in multi‐copy gene families. This innovative tool enabled fast, parallel screening of the samples in this study and facilitated the identification of the specific editing patterns produced by the designed constructs.

Wheat is a staple cereal in the human diet. Despite its significance, an increasing percentage of the population suffers adverse reactions to wheat, which are triggered by wheat gluten, particularly the gliadin fractions. In this study, we employed CRISPR/Cas [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] multiplexing to introduce targeted mutations into γ- and ω-gliadin genes of wheat, to produce lines deficient in one or both immunogenic gliadin fractions simultaneously. For this study, eight single guide RNAs (sgRNAs) were designed and combined into four plasmids to produce 59 modified wheat lines, of which 20 exhibited mutations in the target genes. Characterization of these lines through Sanger sequencing or next-generation sequencing revealed a complex pattern of InDels, including deletions spanning multiple sgRNAs. The mutations were transmitted to the offspring, and the analysis of homozygous derived lines by reverse-phase HPLC and monoclonal antibodies showed a 97.7% reduction in gluten content. Crossing these lines with other CRISPR/Cas lines deficient in the α-gliadins allowed multiple mutations to be combined. This work represents an important step forward in the use of CRISPR/Cas to develop gluten-free wheat.

Drought and heat are the main abiotic constraints affecting durum wheat production. This study aimed to screen for tolerance to drought, heat, and combined stresses in durum wheat, at the juvenile stage under controlled conditions. Five durum wheat genotypes, including four landraces and one improved genotype, were used to test their tolerance to abiotic stress. After 15 days of growing, treatments were applied as three drought levels (100, 50, and 25% field capacity (FC)), three heat stress levels (24, 30, and 35°C), and three combined treatments (100% FC at 24°C, 50% FC at 30°C and 25% FC at 35°C). The screening was performed using a set of morpho-physiological, and biochemical traits. The results showed that the tested stresses significantly affect all measured parameters. The dry matter content (DM) decreased by 37.1% under heat stress (35°C), by 37.3% under severe drought stress (25% FC), and by 53.2% under severe combined stress (25% FC at 35°C). Correlation analyses of drought and heat stress confirmed that aerial part length, dry matter content, hydrogen peroxide content, catalase, and Glutathione peroxidase activities could be efficient screening criteria for both stresses. The principal component analysis (PCA) showed that only the landrace Aouija tolerated the three studied stresses, while Biskri and Hedhba genotypes were tolerant to drought and heat stresses and showed the same sensitivity under combined stress. Nevertheless, improved genotype Karim and the landrace Hmira were the most affected genotypes by drought, against a minimum growth for the Hmira genotype under heat stress. The results showed that combined drought and heat stresses had a more pronounced impact than simple effects. In addition, the tolerance of durum wheat to drought and heat stresses involves several adjustments of morpho-physiological and biochemical responses, which are proportional to the stress intensity.

Gluten proteins are responsible for the wheat breadmaking quality. However, gluten is also related to human pathologies for which the only treatment is a gluten-free diet (GFD). GFD has gained popularity among individuals who want to reduce their gluten intake. Tritordeum is a cereal species that originated after crossing durum wheat with wild barley and differs from bread wheat in its gluten composition. In this work, we have characterized the immunogenic epitopes of tritordeum bread and results from a four-phase study with healthy adults for preferences of bread and alterations in the gut microbiota after consuming wheat bread, gluten-free bread, and tritordeum bread are reported. Tritordeum presented fewer peptides related to gluten proteins, CD-epitopes, and IgE binding sites than bread wheat. Participants rated tritordeum bread higher than gluten-free bread. Gut microbiota analysis revealed that the adherence to a strict GFD involves some minor changes, especially altering the species producing short-chain fatty acids. However, the short-term consumption of tritordeum bread does not induce significant changes in the diversity or community composition of the intestinal microbiota in healthy individuals. Therefore, tritordeum bread could be an alternative for healthy individuals without wheat-related pathologies who want to reduce their gluten consumption without harming their gut health.