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 cis-regulatory elements using transgenic technology, including a female- and fat body-specific promoter, vitellogenin; testis-specific promoters, Radial spoke head 1 (BmR1) and beta-tubulin 4 (Bmβ4). Here we reported a cis-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-1kb, and nos-2kb, respectively) 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 cis-regulatory element was not sex difference. The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and broadly express 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 cis-regulatory element in the early embryo and in the gonad that can be used in applications involving the CRISPR/Cas9 system. This article is protected by copyright. All rights reserved.