A method for authenticating the fidelity of Cryptococcus neoformans knockout collections

Area of Science:

• Microbiology
• Mycology
• Genomics

Background:

• Gene knockout strain collections are essential for understanding gene function in microorganisms.
• Cryptococcus neoformans, a human pathogenic fungus, has a widely used genome-wide deletion collection.
• The accuracy of these collections is critical for reproducible research.

Purpose of the Study:

• To report the discovery of mixed plates from two independent Cryptococcus neoformans knockout libraries.
• To provide a method for authenticating these collections and ensuring correct strain identification.

Main Methods:

• RNA sequencing (RNAseq) to analyze gene expression and identify transcripts.
• Genome sequencing to confirm strain identity.
• Determination of nourseothricin (NAT)-resistance cassette sizes to differentiate libraries.

Main Results:

• The commercial Cryptococcus neoformans knockout collection was found to contain mixed plates from two distinct libraries.
• RNAseq revealed unexpected transcripts and the absence of expected ones, indicating strain misidentification.
• Confirmation of mixed libraries through cassette size analysis and sequencing.

Conclusions:

• The KN99 collection is composed of plates from two independent knockout libraries.
• A simple authentication method is presented for researchers to verify their Cryptococcus neoformans knockout strains.
• Ensuring the accuracy of knockout collections is crucial for advancing fungal research.