Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species.

Ping Zhang,Xudong Zhu,Chenxi Li,Xiaoyu Ma,Yu Wang,Lan Ma

doi:10.3390/jof7070505

Abstract

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

Highlights

The basidiomycete yeasts Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic human pathogens that mainly infect immunocompromised individuals, those with AIDS or who have received organ transplant surgery [1]
In some papers investigating the use of the CRISPR-Cas9 system for C. neoformans, gRNA expression cassettes containing the target sequence were directly synthesized; this is expensive and not feasible for all researchers [23]
The sequencing results revealed that more than 90% of clones randomly selected on selective media successfully introduced the target sequence into the gRNA expression cassette, indicating the high accuracy rate of gRNA construction using the new CRISPR-Cas9 vector

Summary

Introduction

The basidiomycete yeasts Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic human pathogens that mainly infect immunocompromised individuals, those with AIDS or who have received organ transplant surgery [1]. The purified pNK003 backbone fragment, left homologous arm, hph marker cassette, and right homologous arm were mixed and assembled with the in-fusion method to obtain the pNK003/ADE2.E_HR plasmid containing the HR deletion structure, in which gDNA and Cas reside outside the homology arms and are resolved and degraded by double crossover during homologous recombination. This same approach is applicable for the pRH003 plasmid used in the Serotype D strain. The HR deletion structure plasmid could be constructed within one day

Methods

Results

Conclusion