Functional analyses of Pleurotus ostreatus pcc1 and clp1 using CRISPR/Cas9

Boontawon, Tatpong; Nakazawa, Takehito; Horii, Masato; Tsuzuki, Masami; Kawauchi, Moriyuki; Sakamoto, Masahiro; Honda, Yoshio

doi:10.1016/j.fgb.2021.103599

Cited by 17 publications

(18 citation statements)

References 37 publications

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“…Unfortunately, classical breeding is time-consuming and laborious due to the tedious crossing steps [ 7 ]. In contrast, molecular breeding has attracted increasing attention, largely due to its flexibility, accuracy, and high efficiency [ 8 , 9 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

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

Liu

Dong

Liao

et al. 2022

JoF

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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.

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Section: Introductionmentioning

confidence: 99%

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

Liu

Dong

Liao

et al. 2022

JoF

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“…CRISPR/Cas9 system was firstly successfully adapted for the agaricomycete C. cinerea in 2017 ( Sugano et al, 2017 ), and also applied in the edible mushrooms P. ostreatus ( Boontawon et al, 2021a , Boontawon et al, 2021b ). Marker-free genome editing in Pleurotus ostreatus was conducted using transient expression of genes using the CRISPR/Cas9 system ( Koshi et al, 2022 ).…”

Section: Breeding Techniquesmentioning

confidence: 99%

“…The constructed ku70 mutant using the CRISPR/Cas9 system was suitable for target gene insertion and replacement in G. lucidum (Jun-Liang Tu). 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 ).…”

Section: Breeding Techniquesmentioning

confidence: 99%

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

Dong

Miao

Feng

et al. 2022

Current Research in Food Science

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“…However, the lack of efficient genetic engineering tools makes it difficult to improve the physiological characteristics of this species [ 3 ]. Therefore, the development of new strategic approaches, such as genome editing, are being used to overcome this hurdle [ 4 , 5 , 6 ].…”

Section: Introductionmentioning

confidence: 99%

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

Liu

Cui

Wang

et al. 2022

JoF

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CRISPR/Cas9 systems were established in some edible fungi based on in vivo expressed Cas9 and guide RNA. Compared with those systems, the in vitro assembled Cas9 and sgRNA ribonucleoprotein complexes (RNPs) have more advantages, but only a few examples were reported, and the editing efficiency is relatively low. In this study, we developed and optimized a CRISPR/Cas9 genome-editing method based on in vitro assembled ribonucleoprotein complexes in the mushroom Flammulina filiformis. The surfactant Triton X-100 played a critical role in the optimal method, and the targeting efficiency of the genomic editing reached 100% on a selective medium containing 5-FOA. This study is the first to use an RNP complex delivery to establish a CRISPR/Cas9 genome-editing system in F. filiformis. Moreover, compared with other methods, this method avoids the use of any foreign DNA, thus saving time and labor when it comes to plasmid construction.

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Functional analyses of Pleurotus ostreatus pcc1 and clp1 using CRISPR/Cas9

Cited by 17 publications

References 37 publications

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

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

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

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

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