Desenvolvimento e otimização de protocolo para edição de genomas livre de DNA em soja via bombardeamento de ribonucleoproteínas do sistema CRISPR/cas9

• Ano de defesa: 2023
• Autor(a) principal: Ruffo, Gustavo Caséca
• Orientador(a): Sá, Maria Fatima Grossi de
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Católica de Brasília
• Programa de Pós-Graduação: Programa Stricto Sensu em Ciências Genômicas e Biotecnologia
• Departamento: Escola de Exatas, Arquitetura e Meio Ambiente
• País: Brasil
• Palavras-chave em Português: Soja; DNA; Genomas; Agricultura
• Palavras-chave em Inglês: Soybean; DNA; Genome; Agriculture
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS
• Link de acesso: https://bdtd.ucb.br:8443/jspui/handle/tede/3353

Soybean (Glycine max [(L.) Merrill]) is among the principal commodities of modern agriculture, being the main source of vegetal protein and feedstock for biodiesel. In 2022/23 harvest, soybean represented 30% of the farming revenue for Brazil’s GDP, which is the major producer of this crop in the same period, contributing with 150 million of tons (41.6% of annual production). CRISPR/Cas9 genome editing technology has already been used for enhancing yield and quality of soybean through precise and effective modifications in genome. DNA-free methods abstain the target organism in from any contact with exogenous DNA, usually delivering CRISPR/Cas9 components through ribonucleoproteins (RNP) or mRNA. This strategy allows a reduction in cost and market insertion time. In this work, A DNA-free CRISPR/Cas9 genome editing method for soybean from the component synthesis and validation until the in vivo application. Since there is no DNA-free protocol aiming soybean molecular breeding to date. The heterologous expression parameters optimization of Staphylococcus aureus Cas9 (saCas9) was made in a variable funneling model based in a relevance criterion. First, E. coli BL21(DE3), pLysS(DE3) and Rosetta(DE3) strains was transformed with p6XHis-NLS-SaCas9 vector. No detectable expression levels were observed for Rosetta, while BL21 pLysS presented satisfactory SaCas9 production confirmed through Western Blotting. In comparison with LB, TB and SB media enabled an increase in SaCas9 yield, while TB showd the best result with an 8.6-fold enhancement. Also, the amount of SaCas9 in soluble fraction increases with extending the expression time to 20 hours and reducing the temperature from 37°C to 28 and 18°C. A reduction in IPTG concentration from 1 mM resulted in higher yields until a threshold of 0.25 mM, while reducing even more can decrease the Sacas9 production. Whis all optimized expression conditions mentioned, a maximum yield of 25 mg/L was obtained. Posteriorly, sgRNAs were designed with CRISPR-P2.0 software aiming phytoene desaturase (GmPDS) gene, since its silencing causes a reduction in green color of leaves due to a depletion in chloroplasts, thus helping preliminary validation of editing events. 4 sgRNA were selected having compatibility with PAM sequences for SaCas9 and streptococcus pyogenes Cas9 (SpCas9). In vitro activity assays highlighted sgRNA74, with the highest efficiency of 70-90% in both copies of GmPDS in chromosomes 11 and 18. In vivo assays of delivering RNP for embryonic axis through gold particle bombardment. Nevertheless, conditions still need more optimization, including the usage of other explants like embryogenic callus. In conclusion, the protocol in development is a promising alternative for a more rapid and efficient genome editing approach aiming molecular breeding.