Single and Multiplexed Gene Editing in Ustilago maydis Using CRISPR-Cas9

Area of Science:

• Molecular Biology
• Plant Pathology
• Mycology

Background:

• Ustilago maydis is a key model organism for studying biotrophic plant pathogens.
• Understanding gene function in U. maydis is crucial for elucidating plant-pathogen interactions and virulence mechanisms.

Purpose of the Study:

• To provide a detailed protocol for generating CRISPR plasmids for gene editing in U. maydis.
• To outline the steps for creating edited clonal populations and selecting desired mutants.

Main Methods:

• CRISPR plasmid construction for single and multiplexed gene editing.
• Transformation protocols for U. maydis.
• Methods for generating Cas9-free edited clonal populations.
• Selection strategies for identifying desired gene edits.

Main Results:

• Successful generation of CRISPR plasmids for targeted gene modification in U. maydis.
• Establishment of a workflow for producing stable, edited U. maydis strains.
• Demonstration of efficient selection of desired clones post-editing.

Conclusions:

• This protocol facilitates efficient genetic manipulation of U. maydis.
• The described methods will accelerate research into U. maydis virulence and plant-pathogen interactions.
• The protocol is adaptable for various gene editing applications in U. maydis research.