An Intergenic "safe Haven" Region in Cryptococcus Neoformans Serotype D Genomes

Cryptococcus neoformans is an opportunistic human fungal pathogen and serves as a model organism for studies of eukaryotic microbiology and microbial pathogenesis. C. neoformans species complex is classified into serotype A, serotype D, and AD hybrids, which are currently considered different subspecies. Different serotype strains display varied phenotypes, virulence, and gene regulation. Genetic investigation of important pathways is often performed in both serotype A and D reference strains in order to identify diversification or conservation of the interrogated signaling network. Many genetic tools have been developed for C. neoformans serotype A reference strain H99, including the gene free "safe haven" (SH) regions for DNA integration identified based on genomic features. However, no such a genomic safe haven region has been identified in serotype D strains. Here, capitalizing on the available genomic, transcriptomic, and chromatin data, we identified an intergenic region named as SH3 for the serotype D reference strains JEC21 and XL280. We also designed a sgRNA and a vector facilitating any alien gene integration into SH3 through a CRISPR-Cas9 system. We found that gene inserted in this region complemented the corresponding gene deletion mutant. Fluorescent reporter gene inserted in SH3 can also be expressed efficiently. Insertion in SH3 itself did not alter the expression of adjacent genes and did not affect the growth or mating of C. neoformans. Thus, SH3 provides a resource for genetic manipulations in serotype D strains and will facilitate comparative analyses of gene functions in this species complex. In addition, the incorporation of the multi-omic data in our selection of the safe haven region could help similar studies in other organisms.