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 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

scholarly journals Homology-directed repair of a defective glabrous gene in Arabidopsis with Cas9-based gene targeting

2018 ◽  
Author(s):  
Florian Hahn ◽  
Marion Eisenhut ◽  
Otho Mantegazza ◽  
Andreas P.M. Weber
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Strand Break ◽  
Double Strand Break ◽  
In Planta ◽  
End Joining ◽  
Trichome Formation ◽  
Break Site ◽  
Repair Template ◽  
Non Homologous End Joining

ABSTRACTThe CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell’s own repair machinery either by the non-homologous end joining pathway or by homologous recombination. While the first repair mechanism results in random mutations at the double-strand break site, homologous recombination uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome. However, frequencies of double-strand break repair by homologous recombination are very low. In this study, we compared two methods that have been reported to enhance frequencies of homologous recombination in plants. The first method boosts the repair template availability through the formation of viral replicons, the second method makes use of an in planta gene targeting approach. Additionally, we comparatively applied a nickase instead of a nuclease for target strand priming. To allow easy, visual detection of homologous recombination events, we aimed at restoring trichome formation in a glabrous Arabidopsis mutant by repairing a defective glabrous1 gene. Using this efficient visual marker, we were able to regenerate plants repaired by homologous recombination at frequencies of 0.12% using the in planta gene targeting approach, while both approaches using viral replicons did not yield any trichome-bearing plants.

scholarly journals Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

2021 ◽  
Vol 7 (2) ◽  
pp. 149
Author(s):  
Sarah-Maria Wege ◽  
Katharina Gejer ◽  
Fabienne Becker ◽  
Michael Bölker ◽  
Johannes Freitag ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Biology ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Model Organism ◽  
Ustilago Maydis ◽  
Genomic Locus ◽  
Targeted Gene Deletion ◽  
Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

Targeted gene deletion in Brettanomyces bruxellensis with an expression-free CRISPR-Cas9 system

2020 ◽  
Vol 104 (16) ◽  
pp. 7105-7115
Author(s):  
Cristian Varela ◽  
Caroline Bartel ◽  
Cristobal Onetto ◽  
Anthony Borneman
Keyword(s):  
Gene Deletion ◽  
Brettanomyces Bruxellensis ◽  
Targeted Gene Deletion

Targeted gene deletion of Leishmania major genes encoding developmental stage-specific leishmanolysin (GP63)

1998 ◽  
Vol 27 (3) ◽  
pp. 519-530 ◽  
Author(s):  
Phalgun B. Joshi ◽  
David L. Sacks ◽  
Govind Modi ◽  
W. Robert McMaster
Keyword(s):  
Developmental Stage ◽  
Gene Deletion ◽  
Leishmania Major ◽  
Major Genes ◽  
Targeted Gene Deletion ◽  
Genes Encoding

Homology dependence of targeted recombination at the Chinese hamster APRT locus

1994 ◽  
Vol 14 (10) ◽  
pp. 6663-6673
Author(s):  
J B Scheerer ◽  
G M Adair
Keyword(s):  
Homologous Recombination ◽  
Sequence Homology ◽  
Chinese Hamster Ovary ◽  
Target Gene ◽  
Gene Deletion ◽  
Chinese Hamster ◽  
Adenine Phosphoribosyltransferase ◽  
Aprt Gene ◽  
Logarithmic Relationship ◽  
Targeted Recombination

Using simple linear fragments of the Chinese hamster adenine phosphoribosyltransferase (APRT) gene as targeting vectors, we have investigated the homology dependence of targeted recombination at the endogenous APRT locus in Chinese hamster ovary (CHO) cells. We have examined the effects of varying either the overall length of targeting sequence homology or the length of 5' or 3' flanking homology on both the frequency of targeted homologous recombination and the types of recombination events that are obtained. We find an exponential (logarithmic) relationship between length of APRT targeting homology and the frequency of targeted recombination at the CHO APRT locus, with the frequency of targeted recombination dependent upon both the overall length of targeting homology and the length of homology flanking each side of the target gene deletion. Although most of the APRT+ recombinants analyzed reflect simple targeted replacement or conversion of the target gene deletion, a significant fraction appear to have arisen by target gene-templated extension and correction of the targeting fragment sequences. APRT fragments with limited targeting homology flanking one side of the target gene deletion yield proportionately fewer target gene conversion events and proportionately more templated extension and vector correction events than do fragments with more substantial flanking homology.

Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker

2015 ◽  
Vol 468 ◽  
pp. 66-74 ◽  
Author(s):  
Panpan Song ◽  
Sha Liu ◽  
Xuena Guo ◽  
Xuejing Bai ◽  
Xiuping He ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Selectable Marker ◽  
Hansenula Polymorpha

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

Gene deletion suggests a role for Trypanosoma cruzi surface glycoprotein GP72 in the insect and mammalian stages of the life cycle

1993 ◽  
Vol 106 (4) ◽  
pp. 1023-1033 ◽  
Author(s):  
A.R. de Jesus ◽  
R. Cooper ◽  
M. Espinosa ◽  
J.E. Gomes ◽  
E.S. Garcia ◽  
...  
Keyword(s):  
Life Cycle ◽  
Trypanosoma Cruzi ◽  
Gene Deletion ◽  
Surface Glycoprotein ◽  
Triatoma Infestans ◽  
Insect Vector ◽  
Null Mutant ◽  
Apparent Lack ◽  
Targeted Gene Deletion ◽  
Mouse Macrophages

We have explored the biological function of a surface glycoprotein (GP72) of Trypanosoma cruzi by studying a null mutant parasite, generated by targeted gene deletion. GP72 deletion affected parasite morphology in several stages of the life cycle. Insect midgut (epimastigote) forms had a detached flagellum (apomastigote) in the null mutant. The abnormal flagellar phenotype persisted during development of the infective (metacyclic) forms but there was no impairment in the acquisition of complement resistance, sialidase expression or cell infectivity. The GP72 null mutant could efficiently infect and proliferate in mouse macrophages and non-phagocytic L6E9 cells. The mammalian stages of the life cycle also showed major morphological abnormalities. During early subcultures in L6E9 cells, few extracellular fully flagellated forms, expressing markers characteristic of trypomastigotes, were seen. The extracellular population consisted almost exclusively of rounded forms with short flagella (micromastigote), which expressed an amastigote-specific surface marker and no sialidase. The propagation of the parasite was not affected, despite the apparent lack of the trypomastigote forms, which are thought to be primarily responsible for cell invasion. After some subcultures, the extracellular population changed to about equal numbers of micromastigotes and a range of flagellated forms that still did not include true trypomastigotes. Instead, the kinetoplast remained close to the nucleus and the flagellum emerged from the middle of the cell (mesomastigote). Half of the flagellum adhered to the cell body and the remainder was free at the anterior end. In Triatoma infestans, the survival of the mutant was dramatically reduced, suggesting that either GP72 itself, or the altered properties of the flagellum, were critical for establishment in the insect vector.

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