CRISPR/Cas9-mediated somatic mutation of the sex-linked translucent (os) gene in the silkworm, Bombyx mori
2019
Tomihara, K. (The University of Tokyo, Bunkyo-ku, Tokyo (Japan). Graduate School of Agricultural and Life Sciences, Department of Agricultural and Environmental Biology) | Satta, K. | Shimada, T. | Kiuchi, T.
The Z chromosome-linked sex-linked translucent (os) mutation in the silkworm Bombyx mori causes a slightly translucent larval integument that results from the reduction of urate granules in epidermal cells. We previously concluded that mutations in an amino acid transporter gene belonging to the solute carrier family are responsible for the os mutant phenotype by positional cloning and sequence analysis of independent os mutant strains. However, direct evidence using functional analysis was missing. Therefore, we performed somatic mosaic analysis of the os candidate gene using the CRISPR/Cas9 system. An in vitro synthesized single guide RNA (sgRNA) and Cas9 protein were mixed and injected into eggs of the p50T strain. We successfully obtained mosaic larvae in which the epidermis comprised both white and slightly translucent areas. Various mutations had been induced at the target sites of the os gene in the genomic DNA extracted from Gsub(0) adult legs, indicating that the amino acid transporter gene is required for the sufficient accumulation of uric acid in epidermal cells granules. In addition, we searched OS homologous proteins in 24 genome-sequenced lepidopteran and 12 other insects. Phylogenetic analysis showed that the OS protein is conserved in all lepidopteran insects. On the other hand, OS proteins were not found in some insect species, such as Apis mellifera, Drosophila melanogaster, and Tribolium castaneum. It is considered that os genes play an important role in lepidopteran insects for their survival in nature.
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