Journal
FEMS MICROBIOLOGY LETTERS
Volume 368, Issue 13, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/femsle/fnab080
Keywords
agaricomycete; mushroom; pyrG; CRISPR/Cas9; genome editing
Categories
Funding
- KAKENHI [18H02254, 18KK0178, 19K22332]
- JSPS Bilateral Program [JPJSBP] [120208402, 120209920]
- Kyoto University Foundation
- Grants-in-Aid for Scientific Research [18KK0178, 18H02254, 19K22332] Funding Source: KAKEN
Ask authors/readers for more resources
This study successfully utilized Cas9 RNP for gene mutagenesis in Pleurotus ostreatus, generating strains resistant to 5-fluoroorotic acid, and demonstrated its applicability for molecular breeding. Additionally, gene disruption via the Cas9 RNP system was also achieved in wild-type P. ostreatus, overcoming the challenges in traditional gene targeting studies.
Until recently, classical breeding has been used to generate improved commercial mushroom strains; however, classical breeding remains to be laborious and time-consuming. In this study, we performed gene mutagenesis using Cas9 ribonucleoprotein (Cas9 RNP) as a plasmid-free genome editing in Pleurotus ostreatus, which is one of the most economically important cultivated mushrooms. The pre-assembled Cas9/sgRNA targeting pyrG was introduced into protoplasts of a wild-type monokaryotic P. ostreatus strain PC9, which resulted in a generation of strains exhibiting resistance to 5-fluoroorotic acid. Small insertions/deletions at the target site were identified using genomic PCR followed by sequencing. The results showed Cas9 RNP-assisted gene mutagenesis could be applied for the molecular breeding in P. ostreatus and in other edible mushroom strains. Furthermore, gene disruption via split-marker recombination using the Cas9 RNP system was also successfully demonstrated in wild-type P. ostreatus PC9. This method could overcome the disadvantages of NHEJ-deficiency in conventional studies with gene targeting, and also difficulty in gene targeting in various non-model agaricomycetes.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available