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

Abstract

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 report 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) 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 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 cis‐regulatory 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.

Disruption of the ovarian serine protease (Osp) gene causes female sterility in Bombyx mori and Spodoptera litura

“…Two 23‐base pair (bp) sgRNA target sites in exon 10 and exon 11 were selected and are referred to as TS1 and TS2, respectively; the ORF region between the two sites is 667 bp in length. As the activator line, we used the strain pBac[IE1‐EGFP‐Nos‐Cas9] ( Nos‐Cas9 ), which encodes the gene encoding the enhanced green fluorescent protein (EGFP) marker; Cas9 expression is driven by the ubiquitous Nos promoter . The plasmid pBac [IE1‐DsRed2‐U6‐sgRNA] ( U6‐sgRNA ) was constructed to express the sgRNA under control of the silkworm U6 promoter and the DsRed fluorescence marker gene under control of the IE1 promoter.…”

“…As the activator line, we used the strain pBac[IE1-EGFP-Nos-Cas9] (Nos-Cas9), which encodes the gene encoding the enhanced green fluorescent protein (EGFP) marker; Cas9 expression is driven by the ubiquitous Nos promoter. 21,62,63 The plasmid pBac [IE1-DsRed2-U6-sgRNA] (U6-sgRNA) was constructed to express the sgRNA under control of the silkworm U6 promoter and the DsRed fluorescence marker gene under control of the IE1 promoter. The primers used for plasmid construction are listed in Table 1.…”

Functional characterization of the cis‐regulatory region for the vitellogenin gene in Plutella xylostella

“…The core part of the promoter is a short DNA sequence that contains an RNA polymerase II binding site and some other general transcriptional factors such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH, and is located upstream of the transcription start site of the gene (Ye et al ., ). The germline‐, tissue‐, sex‐ and stage‐specific cis ‐regulatory promoter elements have been extensively studied in lepidopteran insects, such as the female‐fat‐body‐specific promoter (Xu et al ., ), eye‐specific promoter (Thomas et al ., ), silk‐gland‐specific promoter (Tomita et al ., ), testis‐specific promoter (Xu et al ., ), nanos promoter (Xu et al ., ) and actin3 promoter (Fatyol et al ., ). The demand for alternative cis ‐regulatory elements is going to increase because of the development of transgenic insects for pest management, and it is necessary to identify more cis ‐regulatory elements (such as stage‐, tissue‐ and sex‐specific ones) for precise and efficient gene manipulation in lepidopteran insects.…”

CRISPR Disruption of BmOvo Resulted in the Failure of Emergence and Affected the Wing and Gonad Development in the Silkworm Bombyx mori

“…The binary transgenic system was established by expressing Cas9 and two sgRNAs in separate plasmids. The plasmid pBac[IE1-EGFP-SV40-nos-Cas9 -SV40] , which constitutively expresses Cas9 under the control of the nos gene promoter, has been described by our lab [34]. To obtain a transgenic plasmid constitutively expressing two BmOvo sgRNAs, two cassettes of U6:sgRNA scaffold:polIII terminator were constructed using the silkworm U6 promoter [35].…”