Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System.

Kyunghun Min,Aaron P Mitchell,Carol A Woolford,Yuichi Ichikawa,Michael J Imperiale

doi:10.1128/msphere.00130-16

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C.albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C.albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C.albicans genetic manipulation.

Highlights

Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals
Transformation of the ade2⌬::ARG4 template together with linearized pADE2-single guide RNA (sgRNA) DNA produced red Argϩ colonies at a high frequency, whereas transformation of the ade2⌬::ARG4 template alone produced only white Argϩ colonies (Fig. 1). These results suggested that the Candida CRISPR system induced the homozygous deletion of the ADE2 open reading frame (ORF) (1.7 kb) and integration of the ARG4 cassette
The abundance of red Argϩ transformants from the cotransformation samples argued that functional expression of CaCAS9 and the sgRNA gene had occurred upon transformation

Summary

Introduction

Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. We show here that the CRISPR-Cas genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. Vyas and colleagues greatly accelerated C. albicans genetic analysis by adapting a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) system to rapidly create C. albicans homozygous mutants [3]. We report here our finding that the CaCas9sgRNA expression construct does not require genomic integration for functional activity This observation led us to hypothesize that transient introduction of CaCAS9 and the sgRNA gene might simplify C. albicans genome editing. We report here the evidence in support of this hypothesis and describe our modifications of the Vyas constructs that enable a transient introduction approach

Methods

Results

Conclusion