Dataset Information

Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus.

ABSTRACT: Pleurotus ostreatus is one of the most commercially produced edible mushrooms worldwide. Improved cultivated strains with more useful traits have been obtained using classical breeding, which is laborious and time-consuming. Here, we attempted efficient gene mutagenesis using plasmid-based CRISPR/Cas9 as the first step for non-genetically modified (non-GM) P. ostreatus generation. Plasmids harboring expression cassettes of Cas9 and different single guide RNAs targeting fcy1 and pyrG were individually transferred into fungal protoplasts of the PC9 strain, which generated some strains exhibiting resistance to 5-fluorocytosine and 5-fluoroorotic acid, respectively. Genomic PCR followed by sequencing revealed small insertions/deletions or insertion of a fragment from the plasmid at the target site in some of the drug-resistant strains. The results demonstrated efficient CRISPR/Cas9-assisted genome editing in P. ostreatus, which could contribute to the molecular breeding of non-GM cultivated strains in the future. Furthermore, a mutation in fcy1 via homology-directed repair using this CRISPR/Cas9 system was also efficiently introduced, which could be applied not only for precise gene disruption, but also for insertions leading to heterologous gene expression in this fungus.

SUBMITTER: Boontawon T

PROVIDER: S-EPMC7897337 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus.

Boontawon Tatpong T Nakazawa Takehito T Inoue Chikako C Osakabe Keishi K Kawauchi Moriyuki M Sakamoto Masahiro M Honda Yoichi Y

AMB Express 20210220 1

Pleurotus ostreatus is one of the most commercially produced edible mushrooms worldwide. Improved cultivated strains with more useful traits have been obtained using classical breeding, which is laborious and time-consuming. Here, we attempted efficient gene mutagenesis using plasmid-based CRISPR/Cas9 as the first step for non-genetically modified (non-GM) P. ostreatus generation. Plasmids harboring expression cassettes of Cas9 and different single guide RNAs targeting fcy1 and pyrG were individ ...[more]

PMID: 33609205

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Project description:CRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava (Manihot esculenta), the Phytoene desaturase (MePDS) gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within MePDS exon 13. After Agrobacterium-mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analyzed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90 to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence analysis revealed that 38/38 (100%) of the plant lines examined carried mutations at the targeted MePDS site, with insertions, deletions, and substitutions recorded. One putatively mono-allelic homozygous line (1/19) was found from cv. 60444, while 1 (1/19) and 4 (4/19) putatively bi-allelic homozygous lines were found in 60444 and TME204, respectively. The remaining plant lines, comprised mostly of the chimeras, were found to be putatively heterozygous. We observed minor (1 bp) nucleotide substitutions and or deletions upstream of the 5' and or downstream of the 3' targeted MePDS region. The data reported demonstrates that CRISPR/Cas9-mediated genome editing of cassava is highly efficient and relatively simple, generating multi-allelic mutations in both cultivars studied. Modification of MePDS described here generates visually detectable mutated events in a relatively short time frame of 6-8 weeks, and does not require sequencing to confirm editing at the target. It therefore provides a valuable platform to facilitate rapid assessment and optimization of CRISPR/Cas9 and other genome-editing technologies in cassava.

Dataset Information

Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus.

Publications

Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus.

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