Author:
Zhang Ping,Wang Yu,Li Chenxi,Ma Xiaoyu,Ma Lan,Zhu Xudong
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.
Funder
National Natural Science Foundation of China
Subject
Plant Science,Ecology, Evolution, Behavior and Systematics,Microbiology (medical)
Reference36 articles.
1. Immunology of infection caused by Cryptococcus neoformans;Wormley;Methods Mol. Med.,2005
2. Deciphering the Model Pathogenic Fungus Cryptococcus Neoformans
3. Cryptococcosis: a model for the understanding of infectious diseases
4. Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation;Edman;Mol. Cell. Biol.,1990
5. Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA
Cited by
7 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献