Transformation of Cryptococcus neoformans by electroporation using a transient CRISPR-Cas9 expression (TRACE) system

Lin, Jianfeng; Fan, Yumeng; Lin, Xiaorong

doi:10.1016/j.fgb.2020.103364

Cited by 39 publications

(47 citation statements)

References 35 publications

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“…The tagged alleles were then introduced into the safe haven ( 55 ) of recipient strains by TRACE ( 52 ). Stable transformants were selected after stability testing with five passages on nonselective medium and further analyzed by diagnostic PCR to confirm the replacement as described previously ( 65 ). All primers and plasmids used in this study are listed in Table S1 .…”

Section: Methodsmentioning

confidence: 99%

A PAS Protein Directs Metabolic Reprogramming during Cryptococcal Adaptation to Hypoxia

Zhao

Lin

2021

mBio

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To aerobic organisms, low oxygen tension (hypoxia) presents a physiological challenge. To cope with such a challenge, metabolic pathways such as those used in energy production have to be adjusted. Many of such metabolic changes are orchestrated by the conserved hypoxia-inducible factors (HIFs) in higher eukaryotes. However, there are no HIF homologs in fungi or protists, and not much is known about conductors that direct hypoxic adaptation in lower eukaryotes. Here, we discovered that the transcription factor Pas2 controls the transcript levels of metabolic genes and consequently rewires metabolism for hypoxia adaptation in the human fungal pathogen Cryptococcus neoformans. Through genetic, proteomic, and biochemical analyses, we demonstrated that Pas2 directly interacts with another transcription factor, Rds2, in regulating cryptococcal hypoxic adaptation. The Pas2/Rds2 complex represents the key transcription regulator of metabolic flexibility. Its regulation of metabolism rewiring between respiration and fermentation is critical to our understanding of the cryptococcal response to low levels of oxygen. IMPORTANCE C. neoformans is the main causative agent of fungal meningitis that is responsible for about 15% of all HIV-related deaths. Although an obligate aerobic fungus, C. neoformans is well adapted to hypoxia conditions that the fungus could encounter in the host or the environment. The sterol regulatory element binding protein (SREBP) is well known for its role in cryptococcal adaptation to hypoxia through its regulation of ergosterol and lipid biosynthesis. The regulation of metabolic reprogramming under hypoxia, however, is largely unknown. Here, we discovered one key regulator, Pas2, that mediates the metabolic response to hypoxia together with another transcription factor, Rds2, in C. neoformans. The findings help define the molecular mechanisms underpinning hypoxia adaptation in this and other lower eukaryotes.

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Section: Methodsmentioning

confidence: 99%

A PAS Protein Directs Metabolic Reprogramming during Cryptococcal Adaptation to Hypoxia

Zhao

Lin

2021

mBio

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“…Due to the observation that electroporation often results in the presence of episomally maintained DNA that rarely integrates into the chromosomes of the cell. Efforts, including the use of the dominant selection markers, the split marker genes, and, recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) technique, were made to increase chromosomal integration efficiency and induce HDR [ 37 , 38 , 39 , 40 , 41 ]. We will discuss these efforts later in the chapter.…”

Section: Transformation Methodsmentioning

confidence: 99%

“…The inclusion of a donor DNA resulted in 70 HDR-mediated gene disruption mutants following transformation [ 66 ]. Fan and colleagues provided further improvements through a transient Cas9 expression system (TRACE) that can be delivered through electroporation [ 39 , 41 ].…”

Section: Application Of Crispr-cas9 Technologymentioning

confidence: 99%

Genetic Transformation in Cryptococcus Species

Wang

2021

JoF

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Genetic transformation plays an imperative role in our understanding of the biology in unicellular yeasts and filamentous fungi, such as Saccharomyces cerevisiae, Aspergillus nidulans, Cryphonectria parasitica, and Magnaporthe oryzae. It also helps to understand the virulence and drug resistance mechanisms of the pathogenic fungus Cryptococcus that causes cryptococcosis in health and immunocompromised individuals. Since the first attempt at DNA transformation in this fungus by Edman in 1992, various methods and techniques have been developed to introduce DNA into this organism and improve the efficiency of homology-mediated gene disruption. There have been many excellent summaries or reviews covering the subject. Here we highlight some of the significant achievements and additional refinements in the genetic transformation of Cryptococcus species.

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“…107,108 These collections were profiled to unveil key regulators of virulence and drug resistance in order to map potential anticryptococcal targets to be exploited for therapeutic development. Finally, the genome editing strategy transient CRISPR-Cas9 coupled with electroporation system (TRACE) was recently developed that dramatically improves the efficiency of targeted mutagenesis in the Cryptococcus genome, 109,110 thus providing great promise for the generation of future mutant collections in this species.…”

Section: Chemical Genomics In Cryptococcusmentioning

confidence: 99%

Advances in fungal chemical genomics for the discovery of new antifungal agents

Xue

Robbins

Cowen

2020

Annals of the New York Academy of Sciences

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Invasive fungal infections have escalated from a rare curiosity to a major cause of human mortality around the globe. This is in part due to a scarcity in the number of antifungal drugs available to combat mycotic disease, making the discovery of novel bioactive compounds and determining their mode of action of utmost importance. The development and application of chemical genomic assays using the model yeast Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of diverse molecules in a living cell. Furthermore, complementary assays are continually being developed in fungal pathogens, most notably Candida albicans and Cryptococcus neoformans, to elucidate compound mechanism of action directly in the pathogen of interest. Collectively, the suite of chemical genetic assays that have been developed in multiple fungal species enables the identification of candidate drug target genes, as well as genes involved in buffering drug target pathways, and genes involved in general cellular responses to small molecules. In this review, we examine current yeast chemical genomic assays and highlight how such resources provide powerful tools that can be utilized to bolster the antifungal pipeline.

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Transformation of Cryptococcus neoformans by electroporation using a transient CRISPR-Cas9 expression (TRACE) system

Cited by 39 publications

References 35 publications

A PAS Protein Directs Metabolic Reprogramming during Cryptococcal Adaptation to Hypoxia

A PAS Protein Directs Metabolic Reprogramming during Cryptococcal Adaptation to Hypoxia

Genetic Transformation in Cryptococcus Species

Advances in fungal chemical genomics for the discovery of new antifungal agents

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