Significance: The use of heterologous systems to express spider silk has become an attractive method. However, achieving cost-effective production and high yields is still challenging. Here, we describe the establishment of a targeted gene replacement system in Bombyx mori to express the major ampullate spidroin-1 gene ( MaSp1 ) from the spider Nephila clavipes . With the aid of transcription activator-like effector nuclease-mediated homology-directed repair, we genetically replaced the silkworm fibroin heavy chain gene with MaSp1 with considerable transformation efficiency, and the chimeric MaSp1 yields reached up to 35.2% wt/wt of cocoon shells in transformed silkworms. The genetically modified silk fiber had significant changes in mechanical properties, with improved extensibility. This system will shed light on the future mass production of new biomaterials, including spider silk.

Manipulation of sex determination pathways in insects provides the basis for a wide spectrum of strategies to benefit agriculture and public health. Furthermore, insects display a remarkable diversity in the genetic pathways that lead to sex differentiation. The silkworm, Bombyx mori, has been cultivated by humans as a beneficial insect for over two millennia, and more recently as a model system for studying lepidopteran genetics and development. Previous studies have identified the B. mori Fem piRNA as the primary female determining factor and BmMasc as its downstream target, while the genetic scenario for male sex determination was still unclear. In the current study, we exploite the transgenic CRISPR/Cas9 system to generate a comprehensive set of knockout mutations in genes BmSxl, Bmtra2, BmImp, BmImp^M, BmPSI and BmMasc, to investigate their roles in silkworm sex determination. Absence of Bmtra2 results in the complete depletion of Bmdsx transcripts, which is the conserved downstream factor in the sex determination pathway, and induces embryonic lethality. Loss of BmImp or BmImp^M function does not affect the sexual differentiation. Mutations in BmPSI and BmMasc genes affect the splicing of Bmdsx and the female reproductive apparatus appeared in the male external genital. Intriguingly, we identify that BmPSI regulates expression of BmMasc, BmImp^M and Bmdsx, supporting the conclusion that it acts as a key auxiliary factor in silkworm male sex determination. The sex determination system extremely diverse among organisms including insects in which even each order occupy a different manner of sex determination. The silkworm, Bombyx mori, is a lepidopteran model insect with economic importance. The mechanism of the silkworm sex determination has been in mystery for a long time until a Fem piRNA was identified as the primary female sex determinator recently. However, genetic and phenotypic proofs are urgently needed to fully exploit the mechanism, especially of the male sex determination. In the current study, we provided comprehensively genetic evidences by generating CRISPR/Cas9-mediated knockout mutations for those genes BmSxl, Bmtra2, BmImp, BmImp^M, BmPSI and BmMasc, which were considered to be involved in insect sex determination. The results showed that mutations of BmSxl, BmImp and BmImp^M had no physiological and morphological effects on the sexual development while Bmtra2 depletion caused Bmdsx splicing disappeared and induced embryonic lethality. Importantly, the BmPSI regulates expression of BmMasc, BmImp^M and Bmdsx, supporting the conclusion that it acts as a key auxiliary factor to regulate the male sex determination in the silkworm.