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

Liu, Jianyu; Cui, Haiyang; Wang, Ruijuan; Xu, Zhen; Yu, Hailong; Song, Chunyan; Huan, Liu; Li, Qiaozhen; Xing, Danrun; Sun, Wei; Zou, Gen; Shang, Xiaodong

doi:10.3390/jof8101000

Cited by 6 publications

(6 citation statements)

References 31 publications

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“…The fruiting stage edible fungi is light sensitive stage, light can either stimulate or inhibit fungal development, so light is an important factor in the growth and development of edible fungi. Our study indicated that the highest average growth rate of stipe diameter, pileus diameter, and stipe length of L. decastes was, respectively, detected at bud stage (DAT 15-Day 25), coralline (31,32). Therefore, the degradation of L. decastes mycelium under R treatment in this study might be due to the lack of blue light irradiation or a decreased expression level of membrane transporter protein in L. decastes caused by monochromatic red light.…”

Section: Figurementioning

confidence: 57%

“…In addition, studies also have shown that several important biosynthetic pathways in mushrooms such as the membrane transport protein synthesis and the amino acid biosynthetic were found inactive under monochromatic red light irradiation. The function of membrane transporters was to perceive external stimuli and transmit signals to cells, maintaining the activity of mycelial ( 31 , 32 ). Therefore, the degradation of L. decastes mycelium under R treatment in this study might be due to the lack of blue light irradiation or a decreased expression level of membrane transporter protein in L. decastes caused by monochromatic red light.…”

Section: Discussionmentioning

confidence: 99%

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The development and nutritional quality of Lyophyllum decastes affected by monochromatic or mixed light provided by light-emitting diode

Chen,

Liu,

Guo

et al. 2024

Front. Nutr.

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Edible fungi has certain photo-sensitivity during the mushroom emergence stage, but there has been few relevant studies on the responses of Lyophyllum decastes to different light quality. L. decastes were planted in growth chambers with different light qualities that were, respectively, white light (CK), monochromatic red light (R), monochromatic blue light (B), mixed red and blue light (RB), and the mixture of far-red and blue light (FrB). The photo-sensitivity of L. decastes was investigated by analyzing the growth characteristics, nutritional quality, extracellular enzymes as well as the light photoreceptor genes in mushroom exposed to different light treatments. The results showed that R led to mycelium degeneration, fungal skin inactivation and failure of primordial formation in L. decastes. The stipe length, stipe diameter, pileus diameter and the weight of fruiting bodies exposed to RB significantly increased by 8.0, 28.7, 18.3, and 58.2% respectively, compared to the control (p < 0.05). B significantly decreased the stipe length and the weight of fruiting body, with a decrease of 8.5 and 20.2% respectively, compared to the control (p < 0.05). Increased color indicators and deepened simulated color were detected in L. decastes pileus treated with B and FrB in relative to the control. Meanwhile, the expression levels of blue photoreceptor genes such as WC-1, WC-2 and Cry-DASH were significantly up-regulated in mushroom exposed to B and FrB (p < 0.05). Additionally, the contents of crude protein and crude polysaccharide in pileus treated with RB were, respectively, increased by 26.5 and 9.4% compared to the control, while those in stipes increased by 5.3 and 58.8%, respectively. Meanwhile, the activities of extracellular enzyme such as cellulase, hemicellulase, laccase, manganese peroxidase, lignin peroxidase and amylase were significant up-regulated in mushroom subjected to RB (p < 0.05), which may promote the degradation of the culture materials. On the whole, the largest volume and weight as well as the highest contents of nutrients were all detected in L. decastes treated with RB. The study provided a theoretical basis for the regulation of light environment in the industrial production of high quality L. decastes.

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Section: Figurementioning

confidence: 57%

Section: Discussionmentioning

confidence: 99%

The development and nutritional quality of Lyophyllum decastes affected by monochromatic or mixed light provided by light-emitting diode

Chen,

Liu,

Guo

et al. 2024

Front. Nutr.

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“…The RNP-based method has been reported for some agaricomycetes, such as S. commune (Jan Vonk et al 2019 ), P. ostreatus (Boontawon et al 2021b ), Dichomitus squalens (Kowalczyk et al 2021 ), F. filiformis (Liu et al 2022b ), C. cinerea (Pareek et al 2022 ), and G. lucidum (Eom et al 2023 ) (Table 1 ). Technically, this protocol can be applied to species lacking a suitable selection marker gene, provided that protoplasts can be generated efficiently.…”

Section: Genetic Toolbox For P Ostreatusmentioning

confidence: 99%

“…Heat shock induces fruiting in S. commune (Ohm et al 2012 ). Blue light is required for both fruiting initiation and maturation (stipe elongation, pileus expansion, and meiosis in basidia) in C. cinerea (Kües and Navarro-González 2015 ; Liu et al 2022a , b , c ) but only for initiation in P. ostreatus . In addition, the shape of the fruiting bodies of P. ostreatus is distinct from that of many other agaricomycetes.…”

Section: Sexual Developmentmentioning

confidence: 99%

Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences

Nakazawa,

Kawauchi,

Otsuka

et al. 2024

Appl Microbiol Biotechnol

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Pleurotus ostreatus, also known as the oyster mushroom, is a popular edible mushroom cultivated worldwide. This review aims to survey recent progress in the molecular genetics of this fungus and demonstrate its potential as a model mushroom for future research. The development of modern molecular genetic techniques and genome sequencing technologies has resulted in breakthroughs in mushroom science. With efficient transformation protocols and multiple selection markers, a powerful toolbox, including techniques such as gene knockout and genome editing, has been developed, and numerous new findings are accumulating in P. ostreatus. These include molecular mechanisms of wood component degradation, sexual development, protein secretion systems, and cell wall structure. Furthermore, these techniques enable the identification of new horizons in enzymology, biochemistry, cell biology, and material science through protein engineering, fluorescence microscopy, and molecular breeding. Key points • Various genetic techniques are available in Pleurotus ostreatus. • P. ostreatus can be used as an alternative model mushroom in genetic analyses. • New frontiers in mushroom science are being developed using the fungus.

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“…The mycelia of F. velutipes monokaryon “Dan3” transformed by the Agrobacterium -mediated method were used to obtain transformants. However, the transformation rate was low, only 6.84% (Liu et al, 2017 ); 5 years later, Liu J. et al ( 2022 ) first established a CRISPR/Cas9 genome-editing system in the mushroom F. velutipes using a fast, accurate, and transgene-free RNP complex delivery technology (Wang P. A. et al, 2022 ), greatly improving the targeting efficiency to 100%. In this study, a CRISPR/Cas9 genome-editing method based on in vitro assembled Cas9 and RNPs was developed, which avoids the use of any exogenous DNA, thus saving time and manpower in plasmid construction.…”

Section: Application Progress Of Crispr/cas9 In Edible Fungimentioning

confidence: 99%

Application progress of CRISPR/Cas9 genome-editing technology in edible fungi

et al. 2023

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Edible fungi are not only delicious but are also rich in nutritional and medicinal value, which is highly sought after by consumers. As the edible fungi industry continues to rapidly advance worldwide, particularly in China, the cultivation of superior and innovative edible fungi strains has become increasingly pivotal. Nevertheless, conventional breeding techniques for edible fungi can be arduous and time-consuming. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) is a powerful tool for molecular breeding due to its ability to mediate high-efficiency and high-precision genome modification, which has been successfully applied to many kinds of edible fungi. In this review, we briefly summarized the working mechanism of the CRISPR/Cas9 system and highlighted the application progress of CRISPR/Cas9-mediated genome-editing technology in edible fungi, including Agaricus bisporus, Ganoderma lucidum, Flammulina filiformis, Ustilago maydis, Pleurotus eryngii, Pleurotus ostreatus, Coprinopsis cinerea, Schizophyllum commune, Cordyceps militaris, and Shiraia bambusicola. Additionally, we discussed the limitations and challenges encountered using CRISPR/Cas9 technology in edible fungi and provided potential solutions. Finally, the applications of CRISPR/Cas9 system for molecular breeding of edible fungi in the future are explored.

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

Cited by 6 publications

References 31 publications

The development and nutritional quality of Lyophyllum decastes affected by monochromatic or mixed light provided by light-emitting diode

The development and nutritional quality of Lyophyllum decastes affected by monochromatic or mixed light provided by light-emitting diode

Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences

Application progress of CRISPR/Cas9 genome-editing technology in edible fungi

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