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

Abstract

Flammulina filiformis, previously known as Asian Flammulina velutipes, is one of the most commercially important edible fungi, with nutritional value and medicinal properties worldwide. However, precision genome editing using CRISPR/Cas9, which is a revolutionary technology and provides a powerful tool for molecular breeding, has not been established in F. filiformis. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting pyrG under the control of the gpd promoter and FfU6 promoter, respectively, were delivered into protoplasts of F. filiformis Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of F. filiformis was identified, and ultimately several pyrG mutants exhibiting 5-fluorooric acid (5-FOA) resistance were obtained. Additionally, diagnostic PCR followed by Sanger sequencing revealed that fragment deletion between the two sgRNA target sites or small insertions and deletions (indels) were introduced in these pyrG mutants through the nonhomologous end joining (NHEJ) pathway, resulting in heritable changes in genomic information. Taken together, this is the first report in which a successful CRISPR/Cas9 genome-editing system based on dual sgRNAs was established in F. filiformis, which broadens the application of this advanced tool in Basidiomycetes.

A Simple and Efficient CRISPR/Cas9 System Using a Ribonucleoprotein Method for Flammulina filiformis

“…Boontawon et al established an efficient CRISPR/Cas9-assisted genome-editing system based on plasmid harboring expression cassettes of Cas9 and different single-guide RNAs in P. ostreatus [ 30 ]. Liu et al reported a CRISPR/Cas9 genome-editing system based on a plasmid delivery strategy in F. filiformis but only two mutants were obtained in their study [ 2 ]. The transformation efficiency of the plasmid method is relatively low, and there are many false positives.…”

“…Notably, the plasmid method used with F. filiformis required up to 28 d for the transformants to grow after PEG-mediated transformation [ 2 ]. However, in our RNP complex method, transformant growth occurred after only 11 d. The reason for this difference is unknown.…”

“…filiformis ) [ 1 ] is a commercially valuable and edible fungus. In recent years, with the development of the F. filiformis industry and increased market demand, generating cultivars with high-yield and improved quality has caused important production issues [ 2 ]. However, the lack of efficient genetic engineering tools makes it difficult to improve the physiological characteristics of this species [ 3 ].…”

“…Therefore, the establishment of editing technology independent from GM or exogenous DNA is beneficial to evade regulation of policy. Moreover, the reported editing efficiency is still very low [ 2 ]. In this study, we used an RNP delivery strategy to develop a CRISPR/Cas9 transformation method in F. filiformis , in which the editing efficiency on the pyrG gene was 100%.…”

The AMP-Activated Protein Kinase (AMPK) Positively Regulates Lysine Biosynthesis Induced by Citric Acid in Flammulina filiformis

“…Ge et al P.M. Wang et al) [ 1 ], also known as golden needle mushroom or winter mushroom, is one of the most produced and consumed edible mushroom species in China [ 2 , 3 ]. It is also a species of great interest in macrofungus research today [ 4 , 5 ]. With the gradual application of industrialized large-scale production of F .…”