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

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

doi:10.3389/fgeed.2023.1241035

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

Front Genome Ed. 2023 Dec 7:5:1241035. doi: 10.3389/fgeed.2023.1241035. eCollection 2023.

Authors

José Hernandes-Lopes^{1

2}, Maísa Siqueira Pinto^{1

2}, Letícia Rios Vieira^{1

2}, Patrícia Brant Monteiro^{1

2}, Sophia V Gerasimova^{1

2

3}, Juliana Vieira Almeida Nonato^{1

2}, Maria Helena Faustinoni Bruno^{1

2}, Alexander Vikhorev⁴, Fernanda Rausch-Fernandes^{1

2

5}, Isabel R Gerhardt^{1

2

5}, Laurens Pauwels^{6

7}, Paulo Arruda^{1

2

8}, Ricardo A Dante^{1

2

5}, Juliana Erika de Carvalho Teixeira Yassitepe^{1

2

5}

Affiliations

¹ Genomics for Climate Change Research Center (GCCRC), Universidade Estadual de Campinas, Campinas, Brazil.
² Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, Brazil.
³ Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
⁴ Frontier Engineering School, Novosibirsk State University, Novosibirsk, Russia.
⁵ Embrapa Agricultura Digital, Campinas, Brazil.
⁶ Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
⁷ VIB Center for Plant Systems Biology, Ghent, Belgium.
⁸ Departamento de Genética, Evolução, Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brazil.

Abstract

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.

Keywords: Agrobacterium; B104; BABY BOOM; CRISPR/Cas9; VIRESCENT YELLOW-LIKE; WUSCHEL; off-target; protoplast.

Grants and funding

This work was funded by grant 2016/23218-0 “Genomics for Climate Change Research Center (GCCRC)” from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). JH-L received a postdoctoral fellowship from EMBRAPA and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (381669/2019-0). MP and LV received postdoctoral fellowships from FAPESP (2022/04929-3 and 2020/10677-1, respectively). JN and MB received technical training fellowships from FAPESP (2022/04930-1 and 2022/04441-0, respectively). PM received a postdoctoral fellowship from CNPq (167867/2022-9). SG is supported by a visiting researcher grant by FAPESP (2022/09418-7). IG is recipient of CNPq productivity fellowship (308896/2020-3).