Cargando…

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

Efficient 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 inciden...

Descripción completa

Detalles Bibliográficos
Autores principales: Jan Vonk, Peter, Escobar, Natalia, Wösten, Han A. B., Lugones, Luis G., Ohm, Robin A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529522/
https://www.ncbi.nlm.nih.gov/pubmed/31113995
http://dx.doi.org/10.1038/s41598-019-44133-2
_version_ 1783420407337975808
author Jan Vonk, Peter
Escobar, Natalia
Wösten, Han A. B.
Lugones, Luis G.
Ohm, Robin A.
author_facet Jan Vonk, Peter
Escobar, Natalia
Wösten, Han A. B.
Lugones, Luis G.
Ohm, Robin A.
author_sort Jan Vonk, Peter
collection PubMed
description Efficient 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 efficiently induce homologous recombination. 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.
format Online
Article
Text
id pubmed-6529522
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-65295222019-05-30 High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins Jan Vonk, Peter Escobar, Natalia Wösten, Han A. B. Lugones, Luis G. Ohm, Robin A. Sci Rep Article Efficient 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 efficiently induce homologous recombination. 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. Nature Publishing Group UK 2019-05-21 /pmc/articles/PMC6529522/ /pubmed/31113995 http://dx.doi.org/10.1038/s41598-019-44133-2 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Jan Vonk, Peter
Escobar, Natalia
Wösten, Han A. B.
Lugones, Luis G.
Ohm, Robin A.
High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins
title High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins
title_full High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins
title_fullStr High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins
title_full_unstemmed High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins
title_short High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins
title_sort high-throughput targeted gene deletion in the model mushroom schizophyllum commune using pre-assembled cas9 ribonucleoproteins
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529522/
https://www.ncbi.nlm.nih.gov/pubmed/31113995
http://dx.doi.org/10.1038/s41598-019-44133-2
work_keys_str_mv AT janvonkpeter highthroughputtargetedgenedeletioninthemodelmushroomschizophyllumcommuneusingpreassembledcas9ribonucleoproteins
AT escobarnatalia highthroughputtargetedgenedeletioninthemodelmushroomschizophyllumcommuneusingpreassembledcas9ribonucleoproteins
AT wostenhanab highthroughputtargetedgenedeletioninthemodelmushroomschizophyllumcommuneusingpreassembledcas9ribonucleoproteins
AT lugonesluisg highthroughputtargetedgenedeletioninthemodelmushroomschizophyllumcommuneusingpreassembledcas9ribonucleoproteins
AT ohmrobina highthroughputtargetedgenedeletioninthemodelmushroomschizophyllumcommuneusingpreassembledcas9ribonucleoproteins