Multiple Applications of a Transient CRISPR-Cas9 Coupled with Electroporation (TRACE) System in the Cryptococcus neoformans Species Complex

is a fungal pathogen that claims hundreds of thousands of lives annually. Targeted genetic manipulation through biolistic transformation in drove the investigation of this clinically important pathogen at the molecular level. Although costly and inefficient, biolistic transformation remains the major method for editing the genome as foreign DNAs introduced by other methods such as electroporation are predominantly not integrated into the genome. Although the majority of DNAs introduced by biolistic transformation are stably inherited, the transformation efficiency and the homologous integration rate (∼1-10%) are low. Here, we developed a nsient RISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with lectroporation (TRACE) system for targeted genetic manipulations in the species complex. This method took advantages of efficient genome integration due to double-strand breaks created at specific sites by the transient CRISPR-Cas9 system and the high transformation efficiency of electroporation. We demonstrated that TRACE can efficiently generate precise single-gene deletion mutants using the locus as an example. This system can also effectively delete multiple genes in a single transformation, as evident by the successful generation of quadruple α Δ Δ Δ Δ mutants. In addition to generating gene deletion mutants, we complemented the Δ mutant by integrating a wild-type allele at the "safe haven" region ( ) via homologous recombination using TRACE. Interestingly, introduced DNAs can be inserted at a designated genetic site without any homologous sequences, opening up numerous other applications. We expect that TRACE, an efficient, versatile, and cost-effective gene editing approach, will greatly accelerate research in this field.