A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans

Abstract

Fungal pathogens are an increasingly important cause of human disease and mortality, and Candida albicans is among the most common causes of fungal disease. Studying this important fungal pathogen requires a comprehensive genetic toolkit to establish how different genetic factors play roles in the biology and virulence of this pathogen. Here, we developed a CRISPR-based genetic regulation platform to achieve targeted repression of C. albicans genes. This CRISPR interference (CRISPRi) technology exploits a nuclease-dead Cas9 protein (dCas9) fused to transcriptional repressors. The dCas9 fusion proteins pair with a guide RNA to target genetic promoter regions and to repress expression from these genes. We demonstrated the functionality of this system for repression in C. albicans and show that we can apply this technology to repress essential genes. Taking the results together, this work presents a new technology for efficient genetic repression in C. albicans, with important applications for genetic analysis in this fungal pathogen.

Development and Application of CRISPR/Cas in Microbial Biotechnology

“…Gilbert et al compared different repressive effector domains, including the KRAB (Krüppel associated box) domain, the WRPW domain and the CS (Chromo Shadow) domain, and found that dCas9-KRAB was the best repressor when targeting to a window of -50 to +300 bp relative to the transcription start site (TSS), or 0–100 bp region just downstream of the TSS ( Figure 3C ; Gilbert et al, 2013 , 2014 ). Another dCas9 fusion domain, Mxi1, a mammalian transcriptional repressor domain that is reported to interact with the histone deacetylase Sin3 homolog in yeast, also showed effective repression in yeast ( Gilbert et al, 2013 ; Jensen et al, 2017 ; Schwartz et al, 2017 ; Geller et al, 2019 ; Wensing et al, 2019 ). In another research, KRAB was fused to RNA-binding domains (COM-KRAB) and achieved similar repression effects when targeting DNA sites overlapped the TSS using a scaffold RNA (scRNA) ( Figure 3D ; Zalatan et al, 2015 ).…”

The CRISPR toolbox in medical mycology: State of the art and perspectives

“…The faster and cheaper genetic manipulation of fungal genome and expression facilitated by CRISPR-Cas9 is likely to have a profound impact on medical mycology. Loss of function, gene activation, and gene inhibition strategies can be scaled up as CRISPR-Cas9 libraries [95][96][97], and large-scale library screens can facilitate the identification of new targets for antifungal therapy, of which there is a desperate need [98].…”

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis

“…Wensing and colleagues also developed an improved dCas9 system fused with codon optimised repressor domain of mammalian repressor Mxi1. The authors established an efficient and scalable CRISPR-interference (CRISPRi) system for C. albicans which when paired with corresponding guide RNA, can allow repression of the desired gene [34].…”