EasyGuide Plasmids Support in Vivo Assembly of gRNAs for CRISPR/ Cas9 Applications in Saccharomyces cerevisiae

Autor(es)

Jacobus, Ana P. ; Barreto, Joneclei A. . ; Bem, Lucas S. de ; Menegon, Yasmine A. . ; Fier, Icaro ; Bueno, Joao G. R. ; Santos, Leandro V. dos ; Gross, Jeferson

• Tipo de documento: Artigo Científico
• Fonte: ACS SYNTHETIC BIOLOGY; v. 11, n. 11, p. 6-pg., 2022-10-18.

Resumo

Most CRISPR/Cas9 applications in yeast rely on a plasmid-based expression of Cas9 and its guide RNA (gRNA) containing a 20-nucleotides (nts) spacer tailored to each genomic target. The lengthy assembly of this customized gRNA requires at least 3-5 days for its precloning in Escherichia coli, purification, validation, and cotransformation with Cas9 into a yeast strain. Here, we constructed a series of 12 EasyGuide plasmids to simplify CRISPR/ Cas9 applications in Saccharomyces cerevisiae. The new vectors provide templates for generating PCR fragments that can assemble up to six functional gRNAs directly into yeasts via homologous recombination between the 20-nts spacers. By dispensing precloning in E. coli, yeast in vivo gRNA assembly significantly reduces the CRISPR/Cas9 experimental workload. A highly efficient yeast genome editing procedure, involving PCR amplification of gRNAs and donors, followed by their transformation into a Cas9-expressing strain, can be easily accomplished through a quick protocol. (AU)

• Processo FAPESP: 17/13972-1 - Avaliação de mutações adaptativas ao estresse do etanol em leveduras
• Beneficiário: Jeferson Gross
• Modalidade de apoio: Auxílio à Pesquisa - Programa BIOEN - Regular
• Processo FAPESP: 17/24453-5 - Rede de colaboração: abordagens genéticas modernas para aumentar a tolerância de leveduras a hidrolisados lignocelulósicos enriquecidos
• Beneficiário: Ana Paula Jacobus
• Modalidade de apoio: Auxílio à Pesquisa - Programa BIOEN - Jovens Pesquisadores
• Processo FAPESP: 20/07918-7 - Abordagens sistêmicas integradas para o desenvolvimento de leveduras como plataformas microbianas em biorrefinarias
• Beneficiário: Leandro Vieira dos Santos
• Modalidade de apoio: Auxílio à Pesquisa - Programa BIOEN - Regular