Gluten-encoding genes have drawn the attention of CRISPR/Cas technology due to their implication in certain human pathologies, such as Celiac Disease (CD), an autoimmune enteropathy in genetically predisposed individuals.

However, the complexity of the genes encoding for gluten proteins poses challenges for developing CRISPR-based gene editing tools, mutation analysis and further selection of edited lines. Particularly, the alpha-gliadin gene family, arranged in multiple copies in tandem across the three wheat subgenomes, has been edited using one single guide RNA (sgRNA) per construct.

Now, we are progressing the approach by multiplexing CRISPR constructs, harboring multiple sgRNAs targeting the same genes. The previous workflow relied on phylogenetic tree construction for characterizing insertions/deletions (InDels) and required human intervention.

It shows the mutation profiles of CRISPR lines and how they are inherited across three generations of plants. Now, we have introduced a new automated pipeline based on reference genes family alignment, enabled by deep amplicon sequencing, and implemented in high-performance computing (HPC) clusters.

This pipeline facilitates the detection of sgRNA-specific InDels edits and covers large deletions using multi-sgRNAs in different sample types. Additionally, next-generation sequencing (NGS)-based analyses provide insights into alpha-gliadins in wild-type lines, allowing the characterization of the natural variation of the family genes and the diversity of their immunogenicity.

Altogether, the integration of bioinformatics tools accelerates the development of low-immunogenic wheat, making faster the screening of edited lines and facilitating the identification and selection of optimal CRISPR targets for this purpose.