Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris

Hyeongju Choi; Sung-Won Park; Junsang Oh; Cheol Soo Kim; Gi-Ho Sung; Hyunkyu Sang

doi:10.1093/femsle/fnad072

Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris

FEMS Microbiol Lett. 2023 Jan 17:370:fnad072. doi: 10.1093/femsle/fnad072.

Authors

Hyeongju Choi¹, Sung-Won Park¹, Junsang Oh², Cheol Soo Kim³, Gi-Ho Sung^{2

4}, Hyunkyu Sang^{1

5}

Affiliations

¹ Department of Integrative Food, Bioscience and Biotechnology, Chonnam National University, Gwangju 61186, Korea.
² Translational Research Division, Biomedical Institute of Mycological Resource, International St. Mary's Hospital, College of Medicine, Catholic Kwandong University, Incheon 22711, Korea.
³ Department of Applied Biology, Chonnam National University, Gwangju 61186, Korea.
⁴ Department of Microbiology, College of Medicine, Catholic Kwandong University, Gangneung 25601, Korea.
⁵ Kumho Life Science Laboratory, Chonnam National University, Gwangju 61186, Korea.

PMID: 37475654
DOI: 10.1093/femsle/fnad072

Abstract

Cordyceps militaris, an entomopathogenic ascomycete, produces edible medicinal mushrooms known to have medicinal and therapeutic functions. To develop the genetic transformation system in C. militaris, green fluorescent protein (GFP) mutants of C. militaris were generated by PEG-mediated protoplast transformation. The CRISPR/Cas9 ribonucleoprotein (RNP) targeting the class III histidine kinase of C. militaris (CmHk1) was then delivered into protoplasts of C. militaris through the transformation system. Mutations induced by the RNP in selected mutants were detected: 1 nt deletion (6 mutants), 3 nt deletion with substitution of 1 nt (1 mutant), insertion of 85 nts (1 mutant), 41 nts (2 mutants), and 35 nts (5 mutants). An in vitro sensitivity assay of the mutants indicated that knockout of CmHk1 reduced sensitivity to two fungicides, iprodione and fludioxonil, but increased sensitivity to osmotic stresses compared to the wild type. Summing up, the CRISPR/Cas9 RNP delivery system was successfully developed, and our results revealed that CmHk1 was involved in the fungicide resistance and osmotic stress in C. militaris.

Keywords: Cordyceps militaris; CRISPR/Cas9; dicarboximide fungicide; histidine kinase; ribonucleoprotein.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems*
Cordyceps* / genetics
Mutation
Ribonucleoproteins / genetics

Substances

Ribonucleoproteins