biostudies-literatureUnknown15(8)Nalley MJR01 AI000272We recently described CRISPR/Cas9-based short homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans, a haploid budding yeast that is the most common cause of fungal meningitis and an emerging model organism. This was achieved by electroporation of strains stably expressing a codon-optimized Cas9 with 2 separate DNA molecules, one encoding a selectable marker flanked by short homology arms and a second encoding a sgRNA under the control of the U6 snRNA promoter. However, the efficiency of desired homology-dependent repair relative to undesired non-homologous end-joining (NHEJ) events can be low and variable. Here, we describe methods and strains enabling extremely efficient (∼99%) homology-dependent genome editing in C. neoformans. This high-efficiency mG3 (Bethesda, Md.)jkaf118https://www.ebi.ac.uk/biostudies/studies/S-EPMC12341884biostudies-literatureNear 100% efficient homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans.PMC12341884Banerjee SNalley MJHuang MYMadhani HDfalseNear 100% efficient homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans.We recently described CRISPR/Cas9-based short homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans, a haploid budding yeast that is the most common cause of fungal meningitis and an emerging model organism. This was achieved by electroporation of strains stably expressing a codon-optimized Cas9 with 2 separate DNA molecules, one encoding a selectable marker flanked by short homology arms and a second encoding a sgRNA under the control of the U6 snRNA promoter. However, the efficiency of desired homology-dependent repair relative to undesired non-homologous end-joining (NHEJ) events can be low and variable. Here, we describe methods and strains enabling extremely efficient (∼99%) homology-dependent genome editing in C. neoformans. This high-efficiency m2025-01-01T00:00:00Z2025 Aug2026-04-07T18:29:47.159Z2026-04-07T16:35:41.391ZS-EPMC123418844046028010.1093/g3journal/jkaf118