Enabling genome editing in tropical maize lines through an improved, morphogenic regulator-assisted transformation protocol

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Hernandes-Lopes, Jose ; Pinto, Maisa Siqueira ; Vieira, Leticia Rios ; Monteiro, Patricia Brant ; Gerasimova, Sophia V. ; Nonato, Juliana Vieira Almeida ; Bruno, Maria Helena Faustinoni ; Vikhorev, Alexander ; Rausch-Fernandes, Fernanda ; Gerhardt, Isabel R. ; Pauwels, Laurens ; Arruda, Paulo ; Dante, Ricardo A. ; Yassitepe, Juliana Erika de Carvalho Teixeira

• Tipo de documento: Artigo Científico
• Fonte: FRONTIERS IN GENOME EDITING; v. 5, p. 14-pg., 2023-12-07.

Resumo

The recalcitrance exhibited by many maize (Zea mays) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve 'universal' transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium-mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER, which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop. (AU)

• Processo FAPESP: 22/09418-7 - Desenvolvimento de uma plataforma para avaliação rápida e eficiente de ferramentas de edição genômica baseadas em CRISPR/Cas em células vegetais
• Beneficiário: Ricardo Augusto Dante
• Modalidade de apoio: Auxílio à Pesquisa - Pesquisador Visitante - Internacional
• Processo FAPESP: 22/04441-0 - Centro de Pesquisa em Genômica Aplicada as Mudanças Climáticas
• Beneficiário: Maria Helena Faustinoni Bruno
• Modalidade de apoio: Bolsas no Brasil - Programa Capacitação - Treinamento Técnico
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• Beneficiário: Maísa de Siqueira Pinto
• Modalidade de apoio: Bolsas no Brasil - Programa Capacitação - Treinamento Técnico
• Processo FAPESP: 22/04930-1 - Cultivo de milho, Introgressão de genes de interesse e Fenotipagem em casa de vegetação
• Beneficiário: Juliana Vieira Almeida Nonato
• Modalidade de apoio: Bolsas no Brasil - Programa Capacitação - Treinamento Técnico
• Processo FAPESP: 16/23218-0 - Centro de Pesquisa em Genômica Aplicada às Mudanças Climáticas
• Beneficiário: Edi Lúcia Sartorato
• Modalidade de apoio: Auxílio à Pesquisa - Programa Centros de Pesquisa em Engenharia
• Processo FAPESP: 20/10677-1 - Transformação genética de milho mediada por Agrobacterium tumefaciens para obtenção de resistência a estresses abióticos
• Beneficiário: Letícia Rios Vieira
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado