StcU-2 Gene Mutation via CRISPR/Cas9 Leads to Misregulation of Spore-Cyst Formation in Ascosphaera apis .

Ascosphaera apis is the causative agent of honey bee chalkbrood disease, and spores are the only known source of infections. Interference with sporulation is therefore a promising way to manage A. apis. The versicolorin reductase gene ( StcU-2 ) is a ketoreductase protein related to sporulation and melanin biosynthesis. To study the StcU-2 gene in ascospore production of A. apis , CRISPR/Cas9 was used, and eight hygromycin B antibiotic-resistant transformants incorporating enhanced green fluorescent protein ( EGFP ) were made and analyzed. PCR amplification, gel electrophoresis, and sequence analysis were used for target gene editing analysis and verification. The CRISPR/Cas9 editing successfully knocked out the StcU-2 gene in A. apis. StcU-2 mutants had shown albino and non-functional spore-cyst development and lost effective sporulation. In conclusion, editing of StcU-2 gene has shown direct relation with sporulation and melanin biosynthesis of A. apis ; this effective sporulation reduction would reduce the spread and pathogenicity of A. apis to managed honey bee. To the best of our knowledge, this is the first time CRISPR/Cas9-mediated gene editing has been efficiently performed in A. apis , a fungal honey bee brood pathogen, which offers a comprehensive set of procedural references that contributes to A. apis gene function studies and consequent control of chalkbrood disease.