Development of a gene-targeting system using CRISPR/Cas9 and utilization of pyrG as a novel selectable marker in Lentinula edodes.

Ayane Kamiya, Hiroki Ueshima, Shota Nishida, Yoichi Honda, Hisatoshi Kamitsuji, Toshitsugu Sato, Haruto Miyamoto, Takuya Sumita, Kosuke Izumitsu, Toshikazu Irie

FEMS microbiology letters 2023 Jan 17

filter terms:

First, we attempted to recombine the Shiitake (Lentinula edodes) pyrG (ura3) gene homologously by introducing a donor vector containing a carboxin resistance gene (lecbxR) flanked by homologous sequences of pyrG into protoplasts of the fungus. However, all the carboxin-resistant transformants only contained ectopic insertions of the exogenous gene and no homologous insertions. Agaricomycetes are generally known for their low efficiency of homologous recombination, and a similar result was shown for L. edodes. We then co-introduced a Cas9 plasmid vector containing a CRISPR/Cas9 expression cassette targeting pyrG and donor plasmid vector. As a result, ∆pyrG strains containing the expected homologous recombination were obtained. However, only two of the seven ∆pyrG strains had the Cas9 sequence; the others did not. Our results suggest that genome editing occurred via the transient expression of the CRISPR/Cas9 cassette in the Cas9 plasmid vector introduced into the fungal cell. Transforming pyrG into a ∆pyrG strain (strain I8) resulted in prototrophic strains with an efficiency of 6.5 strains/experiment. © The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.

Citation

Ayane Kamiya, Hiroki Ueshima, Shota Nishida, Yoichi Honda, Hisatoshi Kamitsuji, Toshitsugu Sato, Haruto Miyamoto, Takuya Sumita, Kosuke Izumitsu, Toshikazu Irie. Development of a gene-targeting system using CRISPR/Cas9 and utilization of pyrG as a novel selectable marker in Lentinula edodes. FEMS microbiology letters. 2023 Jan 17;370