CRISPR-Mediated Endogenous Activation of Fibroin Heavy Chain Gene Triggers Cellular Stress Responses in Embryonic Cells.

The silkworm is an economically important insect, as it is the main producer of silk. Fibroin heavy chain () 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 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 in embryonic (BmE) cells, and the mRNA expression of was successfully activated. In addition, we found that 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 gene. Moreover, the lyso-tracker red and monodansylcadaverine (MDC) staining assays revealed an apparent appearance of autophagy in -activated BmE cells. Therefore, we conclude that the activation of 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 .