A method for authenticating the fidelity of Cryptococcus neoformans knockout collections

Gene knockout strain collections are important tools for discovery in microbiology. Cryptococcus neoformans is the only human pathogenic fungus with an available genome-wide deletion collection, and this resource is widely used by the research community. We uncovered mix-ups in the assembly of the commercially available C. neoformans deletion collection of [~]6,000 unique strains acquired by our lab. While pursuing the characterization of a gene-of-interest, the corresponding deletion strain from the C. neoformans KO collection displayed several interesting phenotypes associated with virulence. However, RNAseq analysis identified transcripts for the putative knockout gene, and the absence of transcripts for a different knockout strain found in the same plate position in an earlier partial knockout collection, raising the possibility that plates from one collection were substituted for the other. This was supported by determining the size of the nourseothricin (NAT)-resistance cassette used to generate the two separate knockout libraries and was confirmed by RNAseq and genome sequencing. Here we report that our KN99 collection is comprised of mixed plates from two independent KO libraries and present a simple authentication method that other investigators can use to distinguish the identities of these KO collections. ImportanceGene knockout strain collections are important tools for discovery in microbiology. Cryptococcus neoformans is the only human pathogenic fungus with an available genome-wide deletion collection, and this resource is widely used by the research community. Here we report that our KN99 collection is comprised of mixed plates from two independent KO libraries and present a simple authentication method that other investigators can use to distinguish the identities of these KO collections. Above all, this article serves as a reminder to 2015 library collection strains before undertaking large phenotyping experiments.