Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system

Abstract

Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species Coprinopsis cinerea. Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter, CcDED1 _pro , with seven times stronger activity in this assay than the conventional promoter GPD2. To develop highly efficient genome editing using CRISPR/Cas9 in C. cinerea, we used the CcDED1 _pro to express Cas9 and a U6-snRNA promoter from C. cinerea to express gRNA. Finally, CRISPR/Cas9-mediated GFP mutagenesis was performed in a stable GFP expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.

Figure 1

Cryopreservation of protoplasts of C. cinerea. Evaluation of protoplasts stored in different buffers at −80 °C for 3 weeks. (a) Schematic image of the protoplast preservation experiments. (b) The buffers used for cryopreservation of protoplasts. (c) Survival rates defined by the number of surviving cells per 10³ preserved cells. Survival rate of fresh protoplasts was defined as 100%. (d) Transformation rates defined by the number of hygromycin-B resistant colonies in 10⁸ preserved cells. Error bars in (b) and (c) show S.D. n = 3; bar plots with the same lower case characters were statistically significant groups as tested by Turkey’s HSD test (P < 0.05). (e) Schematic image of the transient expression assay using preserved protoplasts which were stored at −80 °C with buffer 3 for three weeks. (f) Fluorescent images of protoplasts 24 hrs after transformation. Fresh and cryopreserved protoplasts were compared. Scale bar: 50 µm.

Figure 2

NanoLuc-based luciferase assay using cryopreserved protoplasts identified novel constitutive active promoters in C. cinerea. (a) The flow of luciferase assay in C. cinerea, corresponding data are given in Supplementary Fig. 3. (b) Comparison of three different luciferases driven by AbGPD2pro. Asterisk shows statistical difference calculated with Turkey’s HSD test (P < 0.05). (c) Comparison of activities of various promoters fused with NanoLuc luciferease. Luminescences of NanoLuc were normalized by transformation rate. Bars: S.D., n = 3. (d) Relative luminescence results of the experiments shown in (c), with the luminescence of AbGPD2pro defined as 1. Number ± S.E.

Figure 3

Construction of a CRISPR/Cas9 system in C. cinerea. (a) The CRISPR/Cas9 vector for C. cinerea genome editing. Four types of codon-optimized Cas9 were tested. An NruI site was used for linearization. (b) Capillary gel electrophoresis of the PCR products of CRISPR/Cas9 transformants #17 and #8. Arrowheads show peaks not detected in wild-type. LM; lower marker, UM; upper marker. (c) Sequences obtained by direct sequencing of PCR products from the two CRISPR/Cas9 transformants. The target sequence of gRNA (green) and PAM sequence (yellow) are indicated. The 75-bp insertion in the line #8 is in blue. (d) Comparison of the mutation rates of four types of codon optimization in the transformants as detected by capillary gel electrophoresis analyses.

Figure 4

Isolation of genome-edited mushrooms. (a) Analyses of mosaic rates in the C. cinerea CRISPR/Cas9 mutant lines. Ratios of reads with mutation to total reads are described for each variant. Total read counts were 373 reads for #8 and 621 reads for #17, respectively. (b,c) GFP loss-of-function in the C. cinerea CRISPR/Cas9 mutants. GFP fluorescence was diminished in vegetative mycelium (b), and fruiting body primordia (c) in the transformants. Bars = 1 mm.