Targeted Genome Editing via CRISPR in the Pathogen Cryptococcus neoformans

Abstract

Low rates of homologous integration have hindered molecular genetic studies in Cryptococcus neoformans over the past 20 years, and new tools that facilitate genome manipulation in this important pathogen are greatly needed. To this end, we have investigated the use of a Class 2 CRISPR system in C. neoformans (formerly C. neoformans var. grubii). We first expressed a derivative of the Streptococcus pyogenes Cas9 nuclease in C. neoformans, and showed that it has no effect on growth, production of virulence factors in vitro, or virulence in a murine inhalation model. In proof of principle experiments, we tested the CAS9 construct in combination with multiple self-cleaving guide RNAs targeting the well-characterized phosphoribosylaminoamidazole carboxylase-encoding ADE2 gene. Utilizing combinations of transient and stable expression of our constructs, we revealed that functionality of our CRISPR constructs in C. neoformans is dependent upon the CAS9 construct being stably integrated into the genome, whilst transient expression of the guide RNA is sufficient to enhance rates of homologous recombination in the CAS9 genetic background. Given that the presence of the CRISPR nuclease does not influence virulence in a murine inhalation model, we have successfully demonstrated that this system is compatible with studies of C. neoformans pathogenesis and represents a powerful tool that can be exploited by researchers in the field.

Comparative genomic analysis of Myroides odoratimimus isolates

“…It may be involved in the bacterial evolution, regulation of virulence gene expression, and the enhancement of pathogenicity (Hatoum‐Aslan & Marraffini, ). Particularly, in the pathogen, CRISPR is able to edit genome and modulate gene functions as an adaptive immune system (Arras et al., ; Sontheimer & Barrangou, ). Comparative analysis of the seven strain genomes (PR63039, CCUG10230, CCUG12901, CIP101113, CCUG12700, L41, and H1bi) showed that PR63039 genome contained three types of CRISPRs (Table S1); CCUG10230 genome contained four types of CRISPRs.…”

Genetic Manipulation of a Lipolytic Yeast Candida aaseri SH14 Using CRISPR-Cas9 System

“…The success of the CRISPR-Cas9 system for genome editing of S. cerevisiae led to many studies of the use of the system for genetic manipulation of various industrial or non-conventional yeasts [37][38][39][40]. Especially, Candida spp has attracted considerable interest because it contains several strains showing a significant potential for biotechnological use [41] and clinically important pathogen [42].…”

History of genome editing in yeast

“…In C. neoformans , the expression of Cas9 was achieved by either integrating the Cas9 gene into the genome (Arras et al, ) or delivering it on a linear vector (Wang et al, ). In both cases, the gRNA was introduced into the cells on a linear vector.…”

“…Wang et al () showed that the efficiency of this system to generate nonsense mutations without the donor DNA was up to 80% and with the donor DNA ranged from 20 to 90%. Arras et al () showed that the HR events occur in this fungus with ~70% efficiency using the CRISPR/Cas9 system.…”