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. 2018 Aug 28;115(35):8757-8762.
doi: 10.1073/pnas.1806805115. Epub 2018 Aug 6.

Mass Spider Silk Production Through Targeted Gene Replacement in Bombyx mori

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Free PMC article

Mass Spider Silk Production Through Targeted Gene Replacement in Bombyx mori

Jun Xu et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, Bombyx mori, has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in B. mori by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) in the spider Nephila clavipes We successfully replaced the ∼16-kb endogenous FibH gene with a 1.6-kb MaSp1 gene fused with a 1.1-kb partial FibH sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted MaSp1 integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.

Keywords: Bombyx mori; TALEN; genome editing; spider silk.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic representation of the TALEN-mediated gene replacement system and the targeted integration of transgene constructs. (A) Schematic representation of the FibH gene and the TALEN targeting sites. The thin yellow line represents the FibH genomic locus, with the open boxes signifying the promoter, exons, and poly(A) signal. A 1,166-bp fragment (green box) located at the 5′ end represents the promoter region. The two 42-bp and 15,750-bp fragments (pink boxes) are exon 1 and exon 2, respectively. A 300-bp fragment (gray box) located at the 3′ end is the poly(A) signal. The red and green lightning icons indicate the two TALEN target sites. In the PJET-Red and PJET-MaSp1 donor constructs, a DsRed2 marker expression cassette driven by a baculovirus IE1 promoter was cloned into the PJET-1.2 vector. DNA fragments of 1,356 bp and 1,331 bp at the 5′ and 3′ ends flanking the TALEN sites were PCR-amplified, subcloned into vectors, and used as homologous arms (L-homo and R-homo, respectively). The 1,593-bp MaSp1 partial sequence is shown by the green box. Primer positions for amplification analyses of the integrated insertions in transformed silkworms are shown by arrows. Primer pairs of F1/R1 (or R3) and F2/R2 were used to amplify the 5′- and 3′-end insertion junctions, respectively. (B) Sequencing results of the integrated diagnostic DNA fragments to show 5′ and 3′ junction genome–donor integration.
Fig. 2.
Fig. 2.
Developmental phenotypes of silk glands in WT, MaSp1+/−, and MaSp1+/+ animals. (A) Gross morphology of the silk glands of WT, MaSp1+/−, and MaSp1+/+. The PSGs of MaSp1+/− and MaSp1+/+ were shorter in length than that in the WT animals. Numbers of 1, 2, and 3 indicate the anterior silk gland (ASG), middle silk gland (MSG), and PSG, respectively. (B) The statistical analysis of the length of the PSG (n = 10). Error bar: SD; ** and *** represent significant differences at the 0.01 and 0.001 level (t test) compared with the control. (C) The statistical analysis of the weight of the intact silk gland (n = 10). Error bar: SD; *** represents significant difference at the 0.001 level (t test) compared with the control. (D) Paraffin-embedded sections of the PSG from WT, MaSp1+/−, and MaSp1+/+ animals at the fourth day of the fifth instar stage. Tissues were stained with hematoxylin–eosin and were photographed under a bright field. Blue arrowheads indicate normal internal secretion structures, and green arrowheads indicate the atrophic secretion, which was smaller and vacuolated in MaSp1+/− and MaSp1+/+ animals. (Scale bar: 0.2 mm.) (E) The boundary of the epidermal tissue and secretion examined by the TEM. The yellow arrows show the epidermal tissue of the PSG, and the green arrows show the internal secretion. (Scale bar: 5 μm.)
Fig. 3.
Fig. 3.
MaSp1 expression in silk glands of transformed animals. (A) Relative mRNA expression of FibH determined using qRT-PCR in WT, MaSp1+/−, and MaSp1+/+ animals. ASG, MSG, and PSG were separated for investigation. The results are expressed as the means ± SD of three independent biological replicates. (B) Relative mRNA expression of MaSp1 determined using qRT-PCR in WT, MaSp1+/−, and MaSp1+/+ animals. (C) MaSp1 protein in PSG was subjected to SDS/PAGE analysis followed by Coomassie brilliant blue staining (Left) or Western blotting analysis (Right). Thin band in the WT lane was due to spillover from the large amount of MaSp1 present in the other lanes. The number in the left indicates the protein marker (kilodaltons). The black arrows show the MaSp1 protein.
Fig. 4.
Fig. 4.
Analysis of transformed cocoons. (A) Morphology of the WT-1, FibH+/−, FibH−/−, WT-2, MaSp1+/−, and MaSp1+/+ cocoons. (Scale bar: 1 cm.) WT-1, FibH+/−, and FibH−/− are from the same brood. WT-2, MaSp1+/−, and MaSp1+/+ are from the same brood. (B) The statistical analysis of the weight of the pupa. (C) The statistical analysis of the weight of the cocoon shell. Error bar: SD; n.s. and *** represent significant differences at the nonsignificant and 0.001 level (t test) compared with the control. (D) The internal structure of the silk fiber examined by TEM. The yellow arrows show the sericin layer, and the green arrows represent fibroin. (Scale bars: 2 μm.)
Fig. 5.
Fig. 5.
MaSp1 expression in cocoon shells of transformed animals. (A) Results of Coomassie brilliant blue staining. (B) The results of Western blot using the antibodies for MaSp1 (Left) and FibH (Right). Numbers to the left indicate the protein molecular mass markers (kilodaltons).
Fig. 6.
Fig. 6.
Stress−strain curves of WT and MaSp1+/− silk fibers. The result of the WT fiber is shown in black, and the MaSp1+/− is shown in red respectively.

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References

    1. Tao H, Kaplan DL, Omenetto FG. Silk materials—A road to sustainable high technology. Adv Mater. 2012;24:2824–2837. - PubMed
    1. Tokareva O, Jacobsen M, Buehler M, Wong J, Kaplan DL. Structure-function-property-design interplay in biopolymers: Spider silk. Acta Biomater. 2014;10:1612–1626. - PMC - PubMed
    1. Blamires SJ, Blackledge TA, Tso IM. Physicochemical property variation in spider silk: Ecology, evolution, and synthetic production. Annu Rev Entomol. 2017;62:443–460. - PubMed
    1. Scheibel T. Spider silks: Recombinant synthesis, assembly, spinning, and engineering of synthetic proteins. Microb Cell Fact. 2004;3:14. - PMC - PubMed
    1. Yip EC, Rayor LS. Maternal care and subsocial behaviour in spiders. Biol Rev Camb Philos Soc. 2014;89:427–449. - PubMed

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