Identification of a germline-expression promoter for genome editing in Bombyx mori

Identification of stage- and tissue-specific cis-regulatory elements will enable more precise genomic editing. In previous studies of the silkworm Bombyx mori, we identified and characterized several tissue- and sex-specific cisregulatory elements using transgenic technology, including a female- and fat body-specific promoter, vitellogenin, testis-specific promoters, Radial spoke head 1 (BmRl) and beta-tubulin 4 (Bmp4). Here we report a c/5-regulatory element specific for a somatic and germ cell-expressed promoter, nanos (Bmnos). We investigated activities of three truncated promoter sequences upstream of the transcriptional initiation site sequences of Bmnos in vitro (nos-0.6kb, nos-1 kb and nos-2kb) and in vivo (nos-2kb). In BmN cultured cells, all three lengths drove expression of the gene encoding enhanced green fluorescence protein (EGFP), although nos-2kb had the highest fluorescence activity. In transgenic silkworms, nos-2kb drove EGFP expression at the early embryonic stage, and fluorescence was concentrated in the gonads at later embryonic stages. In addition, this cisregulatory element was not sex differentiated. The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and were broadly expressed in the whole body. The nos-2kb promoter drove the Cas9 system with efficiency comparable to that of the broad-spectrum strong IE1 promoter. These results indicate that Bmnos is an effective endogenous cisregulatory element in the early embryo and in the gonad that can be used in applications involving the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system.