An intergenic “safe haven” region in Cryptococcus neoformans serotype D genomes

Fan, Yumeng; Lin, Xiaorong

doi:10.1016/j.fgb.2020.103464

Cited by 13 publications

(13 citation statements)

References 51 publications

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“…Expression of the inserted gene can be hampered by gene silencing and unstable expression due to position effects, a phenomenon in which the activity of a gene is affected by its location in the genome. To overcome this, gene expression at the GSH was established in some fungi such as C. neoformans, 22,23,38,39 A.fumigatus, 24,25 and Fusarium venenatum. 40 The candidate safe harbor intergenic regions of A. fumigatus and C. neoformans were screened based on the genome annotation and transcriptome data, e.g., SH1 and SH2 of A. fumigatus have convergent configuration with sizes between 1 and 5 kb and with little or no transcript reads, and the TPM values of their neighboring genes are close to the median or average TPM values of the RNA-seq.…”

Section: ■ Discussionmentioning

confidence: 99%

Safe-Harbor-Targeted CRISPR/Cas9 System and Cmhyd1 Overexpression Enhances Disease Resistance in Cordyceps militaris

Liu,

Meng,

Wang

et al. 2023

J. Agric. Food Chem.

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Fungal disease of mushroomCordyceps militaris (CM) caused byCalcarisporium cordycipiticola (CC) is destructive to fruiting body cultivation, resulting in significant economic loss and potential food safety risks. CRISPR/Cas9 genome editing has proven to be a powerful tool for crop improvement but seldom succeeded in mushrooms. Here, the first genomic safe-harbor site, CmSH1 locus, was identified in the CM genome. A safe-harbor-targeted CRISPR/Cas9 system based on an autonomously replicating plasmid was designed to facilitate alien gene integration at the CmSH1 locus. Cmhyd1, one of the hydrophobin genes, was confirmed as a defensive factor against CC infection, and Cmhyd1 overexpression by this system showed enhancement of disease resistance with negligible effect on the agronomic traits of CM. No off-target events and residues of plasmid sequence were tested by PCR and genome resequencing. This study provided the first safe harbor site for genetic manipulations, a safe harbor-targeted CRISPR/Cas9 system, and the first disease-resistant gene-editing breeding system in mushrooms.

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Section: ■ Discussionmentioning

confidence: 99%

Safe-Harbor-Targeted CRISPR/Cas9 System and Cmhyd1 Overexpression Enhances Disease Resistance in Cordyceps militaris

Liu,

Meng,

Wang

et al. 2023

J. Agric. Food Chem.

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“…The most common approach is to inactivate the ku70 or ku80 genes, creating NHEJ-deficient recipient strains that significantly improve the frequency of knockout mutants’ recovery [ 71 , 76 ]. To prevent ectopic integrations during complementation of null mutants from interfering with the virulence and other characteristics of the fungus, some authors have already reported genomic regions considered as safe for genetic integrations, such as the safe haven for C. neoformans [ 77 , 78 ] or RPS10 locus in Candida albicans [ 79 ]. Although we have not yet evaluated a potential safe region for gene reconstitution in Fonsecaea spp., this tool will be of great relevance when it was described.…”

Section: Discussionmentioning

confidence: 99%

Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene

Favilla

Herman

Goersch

et al. 2023

JoF

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Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely reported for those fungi. In this work, we demonstrated that gene deletion and generation of the null mutant by homologous recombination are achievable for Fonsecaea pedrosoi by the use of two approaches: use of double-joint PCR for cassette construction, followed by delivery of the split-marker by biolistic transformation. Through in silico analyses, we identified that F. pedrosoi presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase trpB —which converts chorismate to trp—was disrupted. The ΔtrpB auxotrophic mutant can grow with external trp supply, but germination, viability of conidia, and radial growth are defective compared to the wild-type and reconstituted strains. The use of 5-FAA for selection of trp- phenotypes and for counter-selection of strains carrying the trp gene was also demonstrated. The molecular tools for the functional study of genes, allied to the genetic information from genomic databases, significantly boost our understanding of the biology and pathogenicity of CBM causative agents.

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“…Identification of a well-defined genetic locus for the integration of a transgenic construct is an important step towards establishing a standardized approach for genetic complementation of a gene KO mutant as well as integration of a transgenic construct for various functional genomics studies. Genomic safe-haven sites (also known as genetic safe-harbor sites or the genetic safe-landing sites) have been identified in several organisms including a number of human fungal pathogens (Arras et al 2015; Upadhya et al 2017; Fan and Lin 2020; Pham et al 2020). Recently two safe-haven regions (SH1 and SH2) were described in A. fumigatus located at intergenic regions of chromosome 1 and 2 of the Af293 genome (Pham et al 2020) however no evidence was presented with respect to their potential impact on virulence in a mammalian system.…”

Section: Discussionmentioning

confidence: 99%

“…GSHs are ideal integration sites for basic functional genomics research as well as for gene therapy applications. Similar genetic loci, which are known as “genomic safe-haven sites” (SH), have recently been identified and used successfully in a number of human pathogenic fungi (Arras et al 2015; Upadhya et al 2017; Fan and Lin 2018; Fan and Lin 2020). Recently, two potential genomic safe-haven regions SH1 and SH2 were identified in A. fumigatus based on genomic annotation and transcriptome data profiling (Pham et al 2020).…”

Section: Introductionmentioning

confidence: 99%

Exploring a novel genomic safe-haven site in the human pathogenic mould Aspergillus fumigatus

Furukawa

Rhijn

Chown

et al. 2022

Preprint

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Aspergillus fumigatus is the most important airborne fungal pathogen and allergen of humans ausing high morbidity and mortality worldwide. The factors that govern pathogenicity of this organism are multi-factorial and are poorly understood. Molecular tools to dissect the mechanisms of pathogenicity in A. fumigatus have improved significantly over the last 20 years however many procedures have not been standardised for A. fumigatus. Here, we present a new genomic safe-haven locus at the site of an inactivated transposon, named SH-aft4, which can be used to insert DNA sequences in the genome of this fungus without impacting its phenotype. We show that we are able to effectively express a transgene construct from the SH-aft4 and that natural regulation of promoter function is conserved at this site. Furthermore, the SH-aft4 locus is highly conserved in the genome of a wide range of clinical and environmental isolates including the isolates commonly used by many laboratories CEA10, Af293 and ATCC46645, allowing a wide range of isolates to be manipulated. Our results show that the aft locus can serve as a site for integration of a wide range of genetic constructs to aid functional genomics studies of this important human fungal pathogen.

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An intergenic “safe haven” region in Cryptococcus neoformans serotype D genomes

Cited by 13 publications

References 51 publications

Safe-Harbor-Targeted CRISPR/Cas9 System and Cmhyd1 Overexpression Enhances Disease Resistance in Cordyceps militaris

Safe-Harbor-Targeted CRISPR/Cas9 System and Cmhyd1 Overexpression Enhances Disease Resistance in Cordyceps militaris

Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene

Exploring a novel genomic safe-haven site in the human pathogenic mould Aspergillus fumigatus

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