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

Abstract

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 nsientRISPR (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 quadrupleαΔΔΔΔ 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.

Centromere scission drives chromosome shuffling and reproductive isolation

“…CRISPR transformation experiments were performed as described previously, with minor modifications 70 (Figure S1). Briefly, C. neoformans cells were grown overnight in YPD media and then inoculated into 100 ml of fresh YPD media and grown until OD 600 reached 0.8 to 1.…”

Host carbon dioxide concentration is an independent stress for Cryptococcus neoformans that affects virulence and antifungal susceptibility

“…H99 was labelled with mCherry by integrating plasmid pH3mCHSH2 (Addgene) into the so-called Safe-Haven 2 (SH2) locus described by Upadhya et al (17). Transiently expressed Cas9 (18) was used to generate a double strand break in the SH2 region and pH3mCHSH2, linearized with ApaI, was used as the repair construct. After selection on neomycin containing plates, colonies that remained NEO + and fluorescent were isolated.…”

Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides

“…While the CRISPR/Cas9 technology is now utilized extensively for engineering microbial genomes and editing targeted nucleotide without DSBs, examples remain rare for its application in basidiomycetous yeasts, except the pathogenic yeast Cryptococcus neoformans . Here, we report successful construction of a CRISPR/Cas9 system for genetic manipulation of R. toruloides .…”

“…While the CRISPR/Cas9 technology is now utilized extensively for engineering microbial genomes [19] and editing targeted nucleotide without DSBs, [20] examples remain rare for its application in basidiomycetous yeasts, except the pathogenic yeast Cryptococcus neoformans. [21][22][23][24] Here, we report successful construction of a CRISPR/Cas9 system for genetic manipulation of R. toruloides. This system would facilitate precise manipulation of R. toruloides genome, which will greatly expand our capacity to explore this yeast for production of metabolites of biofuels and nutraceuticals interest.…”