Advances allowing feasible pyrG gene editing by a CRISPR-Cas9 system for the edible mushroom Pleurotus eryngii

Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis

“…However, only the U6 promoter of F. filiformis could successfully screen mutants, while the use of the U6 promoter of G. lucidum failed (data not shown), probably due to unsuccessful transcription of sgRNA. Although a few articles using heterologous promoters reported successful CRISPR/Cas9 genome editing [ 17 ], it seems that most studies regarding CRISPR/Cas9 in Basidiomycete fungi, especially edible and medicinal fungi, tended to adopt their native promoters [ 14 , 39 , 40 ]. Furthermore, we noticed that the U6 promoter of F. filiformis did not contain a TATA box, and a similar phenomenon was also found in G. lucidum [ 39 ], despite the fact that it was vital for U6 transcription in many species [ 29 ].…”

“…However, due to the limited genetic manipulation and transformation methods, there have been relatively few reports describing successful CRISPR systems in edible fungi. For the convenience of screening and verification, most studies attempting to establish CRISPR systems in Basidiomycetes firstly preferred to choose genes that encoded clear morphological phenotypes or physiological properties for editing, such as pyrG in Pleurotus eryngii [ 14 ] and ura3 in Ganoderma lucidum [ 15 ]. Because pyrG / ura3 encodes orotidine-5′-monophosphate decarboxylase, which is not only involved in the key pathway of uracil synthesis, but also can convert 5-fluorooric acid (5-FOA) into the toxic substance 5-fluorouridine, pyrG / ura3 mutants survive and wild-type (WT) die on a medium supplied with uracil and 5-FOA.…”

“…Thus, the RNA polymerase (Pol) II constitutive promoters are adopted to express Cas9, while Pol III promoters such as U6 are suitable for expression of sgRNA, owing to the fact that mRNA processing events such as splicing, 5′ capping, and 3′ poly(A)-tail addition are not involved. Up to now, the gpd (glyceraldehyde-3-phosphate dehydrogenase) or ef3 (elongation factor 3) promoter (Pol II) and U6 promoter (Pol III) have been successfully applied in some edible fungi, including P. eryngii [ 14 ], Pleurotus ostreatus [ 17 ], and Lentinula edodes [ 18 ].…”

Evolutionary Morphogenesis of Sexual Fruiting Bodies in Basidiomycota: Toward a New Evo-Devo Synthesis

“…A promising new source of functional hypotheses are genomic and transcriptomic data sets, especially comparative ones, which can pinpoint dozens to hundreds of genes that show a consistent association with fruiting body development across species. The community has just started to develop and embrace routine genome engineering techniques (CRISPR/Cas9) (317)(318)(319)(320)(321)(322)(323), which will allow us to test functional hypotheses on genes in a systematic and large-scale manner. Population genomics might prove to be another rich source of hypotheses on genotypephenotype mapping, especially in economically interesting species.…”

Transcriptome analysis reveals the potential mechanism of polyethylene packing delaying lignification of Pleurotus eryngii

“…We will continue to observe more packaging materials (nano, edible film) from the transcriptome perspective on the quality of P. eryngii to improve the relevant molecular mechanisms. On this basis, the targeted gene editing strategy may provide a new idea for the improvement of food mushroom varieties ( Kim and Kim, 2016 , Wang et al, 2021 ). Meanwhile, we made a preliminary exploration on the application of CRM to the visualization of lignin cell level in P. eryngii .…”