Genome editing of the fungus Moniliophthora perniciosa with CRISPR/Cas9 ribonucleoproteins.

• Grant number: 25/08089-8
• Support Opportunities: Scholarships in Brazil - Master
• Start date: August 01, 2025
• End date: July 31, 2027
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Paulo José Pereira Lima Teixeira
• Grantee: Nicholas Minomi
• Host Institution: Escola Superior de Agricultura Luiz de Queiroz (ESALQ). Universidade de São Paulo (USP). Piracicaba , SP, Brazil
• Associated research grant: 18/24432-0 - Functional characterization of effector proteins from cacao pathogens and search for their cognate immune receptors, AP.JP

Abstract

The cacao tree (Theobroma cacao) is a perennial plant of great importance to the economy of several countries in Africa, Asia, and Latin America, including Brazil. Cocoa production has been severely impacted by diseases caused by fungi and oomycetes, resulting in significant losses for producers. Among these diseases, witches' broom, caused by the basidiomycete Moniliophthora perniciosa, is the main factor responsible for the decline of Brazilian cacao farming, compromising crop productivity over recent decades. Since the 2000s, advances in the study of this pathogen have been achieved through molecular and genomic approaches, allowing for a better understanding of its biology, including infection mechanisms, pathogenicity, and host interaction. Nevertheless, the lack of an efficient method to generate M. perniciosa mutants still represents a major obstacle for functional investigations. In this context, our group has dedicated efforts to developing genetic transformation protocols for this fungus, making the generation of transgenic strains a routine practice in our laboratory. However, the introduction of the CRISPR/Cas9 system, a highly efficient tool for genome editing, requires the insertion of large DNA cassettes into the fungal genome, which is inefficient and may result in off-target effects due to the continuous production of the Cas9 nuclease. As a complement to these efforts, this project proposes the use of ribonucleoproteins (RNPs), a system that consists of transfecting purified Cas9 enzyme and guide RNA (gRNA) into the cells, without the need for exogenous DNA insertion into the genome or the generation of transgenic strains. This approach offers significant advantages, such as greater control over genome editing, reduced risk of undesired mutations, and elimination of potential toxic effects from prolonged Cas9 expression. The development of this protocol could represent an important advance in the genetic engineering of M. perniciosa, contributing to the understanding of genes involved in the virulence and biology of this pathogen, as well as enabling more effective strategies for managing witches' broom and ensuring the sustainability of Brazilian cacao farming. (AU)