Abstract
Simple SummaryBased on a CRISPRa approach, activating endogenous fibroin heavy chain (FibH) gene expression in Bombyx mori embryonic (BmE) cells, which was driven by a combination of the dCas9-VPR (a tripartite activator, composed of VP64, p65, and Rta) and the sgRNA targeting to the promoter of FibH gene, was performed for investigating the biological roles of FibH in the development of silk gland cells. The activation of the endogenous FibH gene lead to up-regulation of cellular stress responses-related genes, which suggested a significant positive correlation between activated FibH gene expression and cellular stress responses. Moreover, the present findings might provide a potential model for studying the cellular stress responses caused by silk secretion disorder and lay a foundation for the understanding of silk gland development in silk-spinning insects.The silkworm Bombyx mori is an economically important insect, as it is the main producer of silk. Fibroin heavy chain (FibH) gene, encoding the core component of silk protein, is specifically and highly expressed in silk gland cells but not in the other cells. Although the silkworm FibH gene has been well studied in transcriptional regulation, its biological functions in the development of silk gland cells remain elusive. In this study, we constructed a CRISPRa system to activate the endogenous transcription of FibH in Bombyx mori embryonic (BmE) cells, and the mRNA expression of FibH was successfully activated. In addition, we found that FibH expression was increased to a maximum at 60 h after transient transfection of sgRNA/dCas9-VPR at a molar ratio of 9:1. The qRT-PCR analysis showed that the expression levels of cellular stress response-related genes were significantly up-regulated along with activated FibH gene. Moreover, the lyso-tracker red and monodansylcadaverine (MDC) staining assays revealed an apparent appearance of autophagy in FibH-activated BmE cells. Therefore, we conclude that the activation of FibH gene leads to up-regulation of cellular stress responses-related genes in BmE cells, which is essential for understanding silk gland development and the fibroin secretion process in B. mori.
Highlights
IntroductionThe clustered regularly interspaced short palindromic repeats (CRISPR)/Cas was successfully used as a genome editing tool in both Bombyx mori (B. mori) individuals and cultured cells [14,15,16]
We found that deficient Fibroin heavy chain (FibH) perturbed the cellular homeostasis and caused posterior silk gland (PSG) cell atrophy in the fibroin-deficient naked pupa (Nd) mutant [31,32]
The results show that cellular stress responses were significantly triggered in the FibH-activated B. mori embryonic (BmE) cells, which provided novel insight into the understanding of silk gland development and silk secretion process in silkworm B. mori
Summary
The CRISPR/Cas was successfully used as a genome editing tool in both Bombyx mori (B. mori) individuals and cultured cells [14,15,16]. The dCas, a mutant of Cas lacking detectable nuclease activity, has been developed to interfere or activate the gene expression instead of complete knockout, by fusing to a transcriptional repressive or activating domain [17]. The VPR activating domain is a tripartite activator composed of the VP64, p65, and Rta activators, which has been fused to dCas9 [18]. Our previous work indicated that dCas9-VPR is sufficient to activate the expression of target genes in B. mori embryonic (BmE) cells [19]
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