Translucent larval integument and flaccid paralysis caused by genome editing in a gene governing molybdenum cofactor biosynthesis in Bombyx mori

• Published in: Insect biochemistry and molecular biology Vol. 99; pp. 11 - 16
• Main Authors: Fujii, Tsuguru, Yamamoto, Kazunori, Banno, Yutaka
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.08.2018
• Subjects: Animals; Bombyx - genetics; Bombyx - metabolism; Coenzymes - biosynthesis; Coenzymes - genetics; CRISPR/cas9; Gene Editing; Genome editing; Insect Proteins - genetics; Insect Proteins - metabolism; Larva; Metalloproteins - biosynthesis; Metalloproteins - genetics; Molybdenum enzyme; Pteridines; Sulfite oxidase; Uric acid; CRISPR/cas9; Genome editing; Uric acid; Sulfite oxidase; Molybdenum enzyme
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2018.04.008

Translucency of the larval integument in Bombyx mori is caused by a lack of uric acid in the epidermis. Hime'nichi translucent (ohi) is a unique mutation causing intermediate translucency of the larval integument and male-specific flaccid paralysis. To determine the gene associated with the ohi mutation, the ohi locus was mapped to a 400-kb region containing 29 predicted genes. Among the genes in this region, we focused on Bombyx homolog of mammalian Gephyrin (BmGphn), which regulates molybdenum cofactor (MoCo) biosynthesis, because MoCo is indispensable for the activity of xanthine dehydrogenase (XDH), a key enzyme in uric acid biosynthesis. The translucent integument of ohi larvae turned opaque after injection of bovine xanthine oxidase, which is a mammalian equivalent to XDH, indicating that XDH activity is defective in ohi larvae. RT-PCR and sequencing analysis showed that (i) in ohi larvae, expression of the BmGphn gene was repressed in the fat body where uric acid is synthesized, and (ii) there was no amino acid substitution in the ohi mutant allele. Finally, we obtained BmGphn knockout alleles (hereafter denoted as BmGphnΔ) by using CRISPR/Cas9. The resulting ohi/BmGphnΔ larvae had translucent integuments, demonstrating that BmGphn is the gene responsible for the ohi phenotype. Our results show that repressed expression of BmGphn is a causative factor for the defective MoCo biosynthesis and XDH activity observed in ohi larvae. Interestingly, all male BmGphnΔ homozygotes died before pupation and showed a flaccid paralysis phenotype. The genetic and physiological mechanisms underlying this flaccid paralysis phenotype are also discussed. [Display omitted] •The translucent integument of ohi mutants turned opaque following injection with bovine xanthine oxidase.•The expression of Bombyx Gephyrin (BmGphn) mRNA is repressed in ohi mutants.•CRISPR/Cas9-induced mutation in BmGphn resulted in a translucent integument, male-biased flaccid paralysis, and lethality.•BmGphn is essential for the activity of molybdenum enzymes, including xanthine dehydrogenase.•The ohi mutant is molybdenum cofactor deficient.