LAUSR

to adult, there are morphologically striking changes in the pupa regulated by a finely metabolism, these changes include the degradation, remodeling, and neogenesis of tissues. The immobile silkworm pupa live in the cocoon which protects it from unfavorable situations such as desiccation and predation, but, after the completion of metamorphosis the adult moth must escape from its cocoon. At this time, Cocoonase is secreted by which can hydrolyze and soften the cocoon end, then the silk moth can escape from the cocoon after eclosion. This pierced and moth urine stained cocoon cannot be used for reeling, so pupae are killed by heating or the cocoon are stored in the refrigerator before eclosion because the pupal stage is so short (approximately two weeks) to the silk industry. To reduce the labor and energy required for cocoon processing, drying and storage, we expect to knock out the cocoonase gene by CRISPR/CAS9 system for preventing silk moth escaping from cocoon. CRISPR/CAS9 system is a member of gene editing technologies with high specificity and adaptability as well as easy utility, it is the most widely used technology currently. CRISPR/CAS9 system can be used by different methods in silkworm: (1) injecting of mRNA of CAS9 and gRNA into embryo; (2) constructing transgenic lines of CAS9 drove by constitutive promoter or spatio-temporal specific promoter; (3) establishing stably expressed-CAS9 cell lines of silkworm for further study of mechanism at the cellular level. We have constructed cas9 and gRNA (target cocoonase) transgenic line respectively which have been verified in cellular level. The Cocoonase-free silkworm stain can be obtained by crossing two transgenic lines. We expect this silkworm stain can achieve our expectations, meeting production requirements.