Construction of a CRISPR/Cas9-Mediated Genome Editing System in Lentinula edodes

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

“…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 ].…”

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

“…There are three common delivery strategies in the CRISPR-Cas9 genome-editing system: Cas9 nuclease in vivo and sgRNA in vitro (Cas9-expressing chassis with sgRNA in vitro), both Cas9 and sgRNA in vivo (all-in-one plasmid harboring Cas9-expressing cassette and sgRNA cassette), and both Cas9 and sgRNA in vitro (RNP complex) [ 28 ]. In this context, Moon et al successfully disrupted the LeA1 locus of Lentinula edodes by delivering a plasmid containing the LeU6 and LeGPD promoters to express the Cas9 protein [ 29 ]. Wang et al established a CRISPR/Cas9 genome-editing system in G. lucidum based on a plasmid delivery strategy, but the editing efficiency was low [ 14 ].…”

Edible and medicinal fungi breeding techniques, a review: Current status and future prospects

“… Breeding techniques and their characters Test Strains Main results ref Modern breeding techniques CRISPR/Cas9 accurateregulation one or more genes, the characteristics are the same as the traditional mutagenesis; More efficient, accurate and predictable than traditional mutagenesis; Pleurotus ostreatus Marker-free genome editing; 5-FC resistance and Hyg sensitivity strains were isolated; Koshi et al (2022) Pleurotus ostreatus A mutation in fcy1 via homology-directed repair using this CRISPR/Cas9 system was efficiently introduced Boontawon et al, 2021a , Boontawon et al, 2021b Lentinula edodes Transcription of critical mating-related genes was impaired by the CRISPR/Cas9-mediated deletion mutation in the HD1 gene. Moon et al (2021) Ganoderma species Disruption of ura3 gene of both G. lucidum and G. lingzhi was successfully demonstrated Qin et al (2017) Pleurotus eryngii Cloned and introduced a point mutation in an endogenous gene cbxr; a highly efficient pyrG gene editing system has been established in P. eryngii; Wang et al (2021) G. lucidum Significant decrease in the titer of four identified GAs was found in the mutant compared toWT. Wang et al (2020) Schizophyllum commune The use of pre-assembled Cas9 RNPs in a mushroom-forming basidiomycete and may also improve the genetic accessibility of non-model species; Jan Vonk et al (2019) Coprinopsis cinerea Identify key genes regulating processes such as fruiting body develo...…”

“…Functional genes of pcc1 and clp1 from the Pleurotus ostreatus were also studied by the CRISPR/Cas9 knockout system ( Boontawon et al, 2021a , Boontawon et al, 2021b ). The Cas9 system was successfully constructed in the Lentinula edodes for expression of guide RNAs (gRNAs) which could target the mating-type gene HD1 (LeA1) ( Moon et al, 2021 ). The ura3 gene of the two G. lucidum ( G. lucidum 260125 and G. lingzhi ) were successfully disrupted by codon-optimized Cas9 and transcribed gRNA ( Qin et al, 2017 ).…”