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

Peter Jan Vonk,Han A B Wösten,Natalia Escobar,Robin A Ohm,Luis G Lugones

doi:10.1038/s41598-019-44133-2

Peter Jan Vonk, Han A B Wösten + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-44133-2

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Journal: Scientific Reports	Publication Date: May 21, 2019
Citations: 55	License type: open-access

Affiliation: Utrecht University

Abstract

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.

Highlights

Mushrooms are a nutritious food source that can be cultivated on waste streams, making them important for sustainable food production[1]
This endonuclease can be programmed by a ~100 bp single guide RNA to target DNA based on a 20 bp homology sequence, enhancing recombination rates to facilitate insertions, deletions and replacement at the targeted site[27]
Despite repeated attempts no nourseothricin sensitive colonies were identified among over 100 potential mutants, indicating a lack of gene-inactivating mutations. This means that the efficiency of the procedure is less than 1%, which strongly reduces its use in high-throughput gene deletion

Summary

Introduction

Mushrooms are a nutritious food source that can be cultivated on waste streams, making them important for sustainable food production[1]. CRISPR-Cas[9] is a powerful genetic tool greatly enhancing the efficiency of genome editing in a large number of species[27] This endonuclease can be programmed by a ~100 bp single guide RNA (sgRNA) to target DNA based on a 20 bp homology sequence, enhancing recombination rates to facilitate insertions, deletions and replacement at the targeted site[27]. Be expressed using a RNA Polymerase III promoter or transcribed in vitro and co-transformed into the target cell[27,29] This method has been applied to several fungi, including the mushrooms Coprinopsis cinerea and Ganoderma lucidum, where reporter genes have been deleted[17,28]. This procedure requires extensive genetic knowledge of the target species for codon-optimization, expression and sgRNA delivery. In fungi this method has been applied to several yeasts and multicellular ascomycetes, including multiple Aspergillus species, Penicillium chrysogenum and Cryptococcus neoformans[32,33,34,35,36]

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