Genome editing in Ustilago maydis using the CRISPR-Cas system

Area of Science:

• Molecular Biology
• Mycology
• Plant Pathology

Background:

• Ustilago maydis is a significant fungal pathogen affecting maize.
• Efficient genetic tools are crucial for understanding U. maydis virulence mechanisms.
• The CRISPR-Cas9 system offers precise genome editing capabilities.

Purpose of the Study:

• To establish and validate the type II bacterial CRISPR-Cas9 system for gene disruption in Ustilago maydis.
• To develop a transient expression system for CRISPR-Cas9 in U. maydis to minimize off-target effects.
• To facilitate the study of gene function, particularly redundant genes and gene families, in U. maydis virulence.

Main Methods:

• A self-replicating plasmid was constructed to constitutively express a U. maydis codon-optimized cas9 gene and a single guide RNA (sgRNA).
• The sgRNA was placed under the control of the U. maydis U6 small nuclear RNA (snRNA) promoter.
• Transformation and subsequent analysis of progeny were performed to assess gene disruption efficiency and plasmid stability.

Main Results:

• A single-step transformation successfully induced genome editing in U. maydis.
• An average of 70% of the progeny from a single transformant exhibited disruption of the target b gene.
• The self-replicating plasmid was rapidly lost without selection, ensuring transient CRISPR-Cas9 expression.

Conclusions:

• The CRISPR-Cas9 system provides an efficient method for targeted gene disruption in U. maydis.
• Transient expression of CRISPR-Cas9 minimizes potential long-term negative effects of Cas9 activity.
• This technology advances the ability to study gene function and virulence in U. maydis, especially for redundant genes.