Khongthongdam M.Phetruen T.Chanarat S.Mahidol University2025-01-272025-01-272025-01-01Microbiology Spectrum Vol.13 No.1 (2025)https://repository.li.mahidol.ac.th/handle/20.500.14594/103070Cryptococcus neoformans is a globally distributed pathogenic fungus posing a significantthreat to immunocompromised individuals, particularly those with HIV/AIDS. Effectivegenetic manipulation tools are essential for understanding its biology and developing new therapies. However, current genetic tools, including the variation of versatile selectable markers, are limited. This study develops and validates the phosphite dehydrogenase gene (ptxD)/phosphite (Phi) selection system as a non-antibiotic selectable marker for genetic manipulation in C. neoformans. A codon-optimized ptxD gene from Pseudomonas stutzeri was cloned under the TEF promoter. Using the transient CRISPR-Cas9 coupled with electroporation system, we integrated the ptxD gene into the C. neoformans genome and assessed the impact of ptxD integration on cell growth and virulence factors. The ptxD/Phi system effectivelyselected transformed cells on Phi-containing media. Growth assays showed that ptxD integration did not adversely affectcell growth or key virulence factors, including pleomorphism, capsule size, and melanin production. Additionally, we successfully disrupted the ADE2 gene using this system, confirmingits applicability for gene deletion. Taken together, the ptxD/Phi system provides a robust and versatile tool for genetic manipulation in C. neoformans, facilitating further research into its biology and pathogenicity.Environmental ScienceBiochemistry, Genetics and Molecular BiologyMedicineImmunology and MicrobiologyArticleSCOPUS10.1128/spectrum.01618-242-s2.0-852144948682165049739565132