CRISPR/Cas9-mediated inactivation of the soybean agglutinin Le1 gene to improve grain quality

João Matheus Kafer^1,2Alessandra Koltun^2,3Rodrigo Thibes Hoshino^2,4Larissa Girotto⁵Cesar Augusto Silveira²Silvana Regina Rockenbach Marin²Elibio Leopoldo Rech Filho⁶Alexandre Lima Nepomuceno^1,2Liliane Marcia Mertz-Henning^1,2*

• ¹Universidade Estadual de Londrina, Londrina, Brazil
• ²Embrapa Soja, Londrina, Brazil
• ³Universidade Estadual de Maringa, Maringá, Brazil
• ⁴Fundação de Apoio à Pesquisa e ao Desenvolvimento, Sete Lagoas, Brazil
• ⁵Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brasília, Brazil
• ⁶Empresa Brasileira de Pesquisa Agropecuaria Recursos Geneticos e Biotecnologia, Brasília, Brazil

This study applied CRISPR/Cas9 genome editing to inactivate soybean agglutinin, a well-known antinutritional factor in legume seeds, aiming to improve grain digestibility for animal feed. The Le1 gene (Glyma.02G012600), which encodes the major lectin in soybean seeds, was specifically targeted in the soybean cultivar BRS 537. To achieve this, a binary vector delivered via Agrobacterium tumefaciens transformation method was employed, carrying two gRNAs that generated 20 independent events and achieved an editing efficiency of 10%. Among these, the edited event AF12-13-1 harbored a 4-bp deletion in Le1, resulting in a truncated and unstable protein. Loss of lectin functionality was confirmed through complementary biochemical assays: SDS-PAGE showed the absence of the ~30 kDa lectin band, and hemagglutination tests indicated no agglutination activity, consistent with disruption of carbohydrate-binding capacity. Importantly, the mutation was stably inherited, and transgene-free T2 plants lacking sequences of the editing machinery (Bar, Cas9, AtU6 promoter) were identified. Agronomic evaluation further showed that the edited lines maintained agronomic performance comparable to the wild-type cultivar, with no significant differences in yield or thousand-seed weight, indicating that key agronomic traits were preserved. These results demonstrate that Le1 editing is an effective approach to develop soybean cultivars with enhanced nutritional value and digestibility without compromising essential agronomic traits. Improved digestibility can enhance nutrient absorption, optimize feed efficiency, and ultimately support better weight gain and overall productivity in monogastric animals.