scholarly journals High-Throughput Construction of Gene Deletion Cassettes for Generation of Neurospora crassa Knockout Strains

2010 ◽  
pp. 33-40 ◽  
Author(s):  
Patrick D. Collopy ◽  
Hildur V. Colot ◽  
Gyungsoon Park ◽  
Carol Ringelberg ◽  
Christopher M. Crew ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
High Throughput ◽  
Gene Deletion

Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

2013 ◽  
Vol 52 (3) ◽  
pp. 184-189 ◽  
Author(s):  
Weihua Wu ◽  
Amanda Hildebrand ◽  
Takao Kasuga ◽  
Xiaochao Xiong ◽  
Zhiliang Fan
Keyword(s):  
Neurospora Crassa ◽  
Gene Deletion ◽  
Beta Glucosidase

scholarly journals High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins

2019 ◽  
Author(s):  
Peter Jan Vonk ◽  
Natalia Escobar ◽  
Han A. B. Wösten ◽  
Luis G. Lugones ◽  
Robin A. Ohm
Keyword(s):  
Homologous Recombination ◽  
High Throughput ◽  
Gene Deletion ◽  
Selectable Marker ◽  
Schizophyllum Commune ◽  
Model Systems ◽  
Targeted Gene Deletion ◽  
Repair Template ◽  
Low Incidence ◽  
Non Homologous End Joining

AbstractEfficient gene deletion methods are essential for the high-throughput study of gene function. Compared to most ascomycete model systems, gene deletion is more laborious in mushroom-forming basidiomycetes due to the relatively low incidence of homologous recombination (HR) and relatively high incidence of non-homologous end-joining (NHEJ). Here, we describe the use of pre-assembled Cas9-sgRNA ribonucleoproteins (RNPs) to efficiently delete the homeodomain transcription factor gene hom2 in the mushroom-forming basidiomycete Schizophyllum commune by replacing it with a selectable marker. All components (Cas9 protein, sgRNA, and repair template with selectable marker) were supplied to wild type protoplasts by PEG-mediated transformation, abolishing the need to optimize the expression of cas9 and sgRNAs. A Δku80 background further increased the efficiency of gene deletion. A repair template with homology arms of 250 bp was sufficient to induce homologous recombination, whereas 100 bp was not. This is the first report of the use of pre-assembled Cas9 RNPs in a mushroom-forming basidiomycete and this approach may also improve the genetic accessibility of non-model species.

scholarly journals High-Throughput Production of Gene Replacement Mutants in Neurospora crassa

2011 ◽  
pp. 179-189 ◽  
Author(s):  
Gyungsoon Park ◽  
Hildur V. Colot ◽  
Patrick D. Collopy ◽  
Svetlana Krystofova ◽  
Christopher Crew ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
High Throughput ◽  
Gene Replacement

scholarly journals Characterization of Single Gene Deletion Mutants Affecting Alternative Oxidase Production in Neurospora crassa: Role of the yvh1 Gene

2020 ◽  
Vol 8 (8) ◽  
pp. 1186
Author(s):  
Adrien Beau Desaulniers ◽  
Nishka Kishore ◽  
Kelly Adames ◽  
Frank E. Nargang
Keyword(s):  
Neurospora Crassa ◽  
Gene Deletion ◽  
Alternative Oxidase ◽  
Single Gene ◽  
Ribosome Assembly ◽  
Mitochondrial Translation ◽  
Multiple Phenotypes ◽  
Transport Chain ◽  
Zinc Binding Domain ◽  
Single Gene Deletion

The Neurospora crassa AOD1 protein is a mitochondrial alternative oxidase that passes electrons directly from ubiquinol to oxygen. The enzyme is encoded by the nuclear aod-1 gene and is produced when the standard electron transport chain is inhibited. We previously identified eleven strains in the N. crassa single gene deletion library that were severely deficient in their ability to produce AOD1 when grown in the presence of chloramphenicol, an inhibitor of mitochondrial translation that is known to induce the enzyme. Three mutants affected previously characterized genes. In this report we examined the remaining mutants and found that the deficiency of AOD1 was due to secondary mutations in all but two of the strains. One of the authentic mutants contained a deletion of the yvh1 gene and was found to have a deficiency of aod-1 transcripts. The YVH1 protein localized to the nucleus and a post mitochondrial pellet from the cytoplasm. A zinc binding domain in the protein was required for rescue of the AOD1 deficiency. In other organisms YVH1 is required for ribosome assembly and mutants have multiple phenotypes. Lack of YVH1 in N. crassa likely also affects ribosome assembly leading to phenotypes that include altered regulation of AOD1 production.

scholarly journals Towards a Systems Approach in the Genetic Analysis of Archaea: Accelerating Mutant Construction and Phenotypic Analysis inHaloferax volcanii

Archaea ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Ian K. Blaby ◽  
Gabriela Phillips ◽  
Crysten E. Blaby-Haas ◽  
Kevin S. Gulig ◽  
Basma El Yacoubi ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Deletion ◽  
Systems Approach ◽  
Essential Gene ◽  
Gene Knockout ◽  
Deletion Mutants ◽  
Mutant Library ◽  
Phenotypic Analysis ◽  
Genome Wide ◽  
A Genome

With the availability of a genome sequence and increasingly sophisticated genetic tools,Haloferax volcaniiis becoming a model for both Archaea and halophiles. In order forH. volcaniito reach a status equivalent toEscherichia coli, Bacillus subtilis, orSaccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22H. volcaniideletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of anH. volcaniimutant library and suggest that they should form the basis of a larger genome-wide experiment.

scholarly journals Whole Wiskott-Aldrich syndrome protein gene deletion identified by high throughput sequencing

2017 ◽  
Vol 16 (5) ◽  
pp. 6526-6531 ◽  
Author(s):  
Xiangling He ◽  
Runying Zou ◽  
Bing Zhang ◽  
Yalan You ◽  
Yang Yang ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Deletion ◽  
High Throughput Sequencing ◽  
Protein Gene ◽  
Wiskott Aldrich Syndrome ◽  
Syndrome Protein

scholarly journals High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Peter Jan Vonk ◽  
Natalia Escobar ◽  
Han A. B. Wösten ◽  
Luis G. Lugones ◽  
Robin A. Ohm
Keyword(s):  
High Throughput ◽  
Gene Deletion ◽  
Schizophyllum Commune ◽  
Targeted Gene Deletion
Sign in / Sign up

Export Citation Format

Share Document