Optimization of a CRISPR-Cas9 in vitro Protocol for Targeting the SIX9 Gene of Fusarium oxysporum f.sp. cubense race 1 Associated with Banana Fusarium Wilt

Efren Santos-Ordóñez ^* Liliana Villao Jeffrey Vargas Perez Nardy Diez Garcia Freddy Magdama

• ESPOL Polytechnic University, Guayaquil, Ecuador

Fusarium wilt of bananas (Musa spp.), a threat to sustainable banana production worldwide, necessitates immediate action to control the disease. The current strategies are centered on preventing its spread or developing resistant varieties. However, very little is known about the genetic machinery used by the fungus to infect and kill banana plants. Therefore, research the also the focused in understanding the plant-pathogen molecular interaction that could lead also the focus in understanding the plant-pathogen molecular interaction that could lead also focused on understanding the plantpathogen molecular interaction that could lead is focused on understanding the plant-pathogen molecular interaction that could also leads to target genes for knock-out in Fusarium. This study aims to standardize a gene editing protocol using CRISPR Cas9 technology in Fusarium oxysporum f.sp.cubense race 1 (Foc1); specifically, to induce targeted mutations on a particular effector gene, SIX9, of Fusarium oxysporum f.sp. cubense was targeted. This protocol will support future studies on banana-Fusarium interaction by identifying candidate genes for disease resistance for the plant or lack of virulence for the pathogen, by establishing the function of SIX effector proteins and evaluating the fungus's infection capacity through pathogenicity assays. These findings are crucial and potentially game-changing for the global banana industry. Once established, the function of SIX effector proteins could revolutionize our understanding of the disease and pave the way for more effective control strategies.