A method for authenticating the fidelity of Cryptococcus neoformans knockout collections

Area of Science:

• Microbiology
• Mycology
• Genetics

Background:

• Gene knockout (KO) strain collections are essential for microbiological research.
• The genome-wide deletion collection for *Cryptococcus neoformans*, a significant human fungal pathogen, is widely utilized.
• Accurate strain identification is critical for the reliability of experiments using KO collections.

Purpose of the Study:

• To identify and rectify mix-ups within the *Cryptococcus neoformans* gene knockout strain collection.
• To develop a rapid and reliable method for authenticating KO strains.
• To alert researchers to potential inaccuracies in existing KO libraries.

Main Methods:

• RNA sequencing (RNAseq) to detect unexpected gene transcripts.
• Genome sequencing to confirm strain identity.
• Development of a PCR-based assay targeting size differences in nourseothricin (NAT)-resistance cassettes.

Main Results:

• Mix-ups were identified in approximately nine of the first 15 plates of the KN99ɑ KO collection.
• The PCR assay effectively distinguished between two different KO collections based on NAT-resistance cassette size.
• Genome sequencing confirmed the presence of incorrect strains due to plate swaps.

Conclusions:

• The commercially available *C. neoformans* KO collection contains mixed plates from different libraries.
• A simple PCR authentication method can identify these mix-ups.
• Researchers using the 2015 KO library collection should screen plates before large-scale experiments to ensure accuracy.