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

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 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.

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

“…The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been successfully applied to genome editing in animals, plants and microbes (Arazoe et al, 2015;Bortesi et al, 2016;Li et al, 2019;Schultz et al, 2019). Recently, the CRISPR/Cas9 system has been used for the disruption of genes in higher fungi such as Coprinopsis cinerea, Cordyceps militaris and Schizophyllum commune (Sugano et al, 2017;Chen et al, 2018;Vonk et al, 2019). Qin et al reported the disruption of the ura3 gene of G. lucidum and G. lingzhi, which revealed the potential of the CRISPR/Cas9 system for disrupting the genes of Ganoderma (Qin et al, 2017).…”

Transcriptome data reveal conserved patterns of fruiting body development and response to heat stress in the mushroom-forming fungus Flammulina filiformis

“…Studies on these two model species using tools such as DNA-mediated transformation, RNA interference, and CRISPR/Cas9 etc. have pioneered our knowledge of the multicellular development, mating pheromone, and receptor signaling pathways in the Agaricomycetes [14][15][16][17][18]. More recently, studies also focused on ecologically or economically important non-model species, which included the saprotrophic fungi (Agaricus bisporus, Flammulina filiformis, Lentinula edodes, Lentinus tigrinus, Cyclocybe aegerita), plant pathogen (Armillaria ostoyae), and the ectomycorrhizal fungi (Laccaria bicolor) [2,[19][20][21][22][23][24][25][26].…”

The transcription factor Roc1 is a regulator of cellulose degradation in the wood-decaying mushroom Schizophyllum commune

“…The sgRNAs were designed on regions downstream and upstream of the up-flank and downflank of roc1, respectively, and one sgRNA was selected for each region as previously described 17 .…”

“…In general, very little is known about the regulatory mechanisms involved in plant biomass degradation in the basidiomycetes.Schizophyllum commune is a model system for mushroom-forming fungi in the class Agaricomycetes. Several molecular tools have been developed for this organism, including an efficient gene deletion protocol 17,18 and a ChIP-Seq protocol for Histone H3 to study the epigenetic landscape 19 .S. commune has a wide geographical distribution and is generally found as white fruiting bodies growing on wood.…”

“…Schizophyllum commune is a model system for mushroom-forming fungi in the class Agaricomycetes. Several molecular tools have been developed for this organism, including an efficient gene deletion protocol 17,18 and a ChIP-Seq protocol for Histone H3 to study the epigenetic landscape 19 .…”