Establishment and application of a silkworm CRISPR/Cas9 tool for conditionally manipulating gene disruption in the...

Spatial or temporal specific gene knockout system is a valuable tool for studying the molecular mechanisms underlying developmental processes. The integument is essential for insect fitness and survival, but tools for dissecting function of genes in this tissue are lacking. In this study, we firstly identified an epidermis specifically expressed gene of the domesticated silkworm, BmCPG25, by comparative transcriptomic analysis. Furthermore, a transgenic silkworm expressing the RNA dependent CRISPR-Cas9 protein driven by the regulatory region of the BmCPG25 was established. Immunochemistry analysis showed the endonuclease was specifically expressed in the nuclear of epidermal cells. We also validated the efficiency of this system by disrupting the function of an epidermis specifically expressed cuticular protein gene (Cpr21) and a ubiquitously expressed cuticular gene (Cph18), respectively. In summary, we successfully constructed a conditional knockout toolkit to manipulate the gene editing in epidermal cells, which provides a valuable approach to study the molecular mechanism of integument development.