Feb 16, 2018

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

Genetics
Yumeng Fan, Xiaorong Lin

Abstract

Cryptococcus neoformans is a fungal pathogen that claims hundreds of thousands of lives annually. Targeted genetic manipulation through biolistic transformation in C. neoformans 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 Cryptococcus 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 Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation (TRACE) system for targeted genetic manipulations in the C. neoformans 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 ADE2 locus as an example. This system can al...Continue Reading

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Mentioned in this Paper

Transformation, Genetic
Shuttle Vectors
Research
Infection by Cryptococcus Neoformans
Cryptococcus <Tremellales>
Genome
Genes
Biolistics
CRISPR-Cas Systems
Gene Editing

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