Genome editing of BmFib-H gene provides an empty Bombyx mori silk gland for a highly efficient bioreactor

Abstract

Evolution has produced some remarkable creatures, of which silk gland is a fascinating organ that exists in a variety of insects and almost half of the 34,000 spider species. The impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to 25%) make the silk gland of Bombyx mori hold great promise to be a cost-effective platform for production of recombinant proteins. However, the extremely low production yields of the numerous reported expression systems greatly hindered the exploration and application of silk gland bioreactors. Using customized zinc finger nucleases (ZFN), we successfully performed genome editing of Bmfib-H gene, which encodes the largest and most abundant silk protein, in B. mori with efficiency higher than any previously reported. The resulted Bmfib-H knocked-out B. mori showed a smaller and empty silk gland, abnormally developed posterior silk gland cells, an extremely thin cocoon that contain only sericin proteins, and a slightly heavier pupae. We also showed that removal of endogenous Bmfib-H protein could significantly increase the expression level of exogenous protein. Furthermore, we demonstrated that the bioreactor is suitable for large scale production of protein-based materials.

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial

“…A previous study has shown that silkworm PSGs that express Bcl-2-associated X protein become ablated (17), although this study did not present information on the cocoons and the development of the manipulated individual after pupation. Furthermore, a separate report describing T7 RNA polymerase-modified PSGs of transgenic silkworms showed decreased but detectable FibL and FibH expression (33), whereas another study showed that silkworms with transcription activatorlike effector nuclease-mediated FibH gene disruption produced thin-layered cocoons containing no FibH but a normal level of FibL (34). The Nd silkworm strain has a natural genetic mutation that affects silk glands and produces small quantities of cocoons that have very high amounts of sericin and varying percentages of fibroin (22).…”

Comparative proteomic analysis of silkworm fat body after knocking out fibroin heavy chain gene: a novel insight into cross-talk between tissues

“…Silkworms used in this study, including BmFib-H gene knockout B. mori line (FGKO) and wide-type Dazao, were kindly provided by Dr. Sanyuan Ma at the State Key Laboratory of Silkworm Genome Biology, Southwest University of China (Ma et al 2014). Silkworms were reared under normal conditions at 25°C with fresh mulberry leaves.…”

“…Knocking out BmFib-H gene Electronic supplementary material The online version of this article (doi:10.1007/s10142-015-0461-0) contains supplementary material, which is available to authorized users. by genome editing using customized ZFN (Ma et al 2014) results in smaller size of both posterior silk gland (PSG) and PSG cells, reduced fibroin production, and thin cocoons with few sericins and some small fibroin proteins. Interestingly, the pupal body weight, which is not directly related to the BmFib-H gene, is significantly increased compared to that of wild-type Dazao (Chen et al 2015;Ma et al 2014).…”

“…by genome editing using customized ZFN (Ma et al 2014) results in smaller size of both posterior silk gland (PSG) and PSG cells, reduced fibroin production, and thin cocoons with few sericins and some small fibroin proteins. Interestingly, the pupal body weight, which is not directly related to the BmFib-H gene, is significantly increased compared to that of wild-type Dazao (Chen et al 2015;Ma et al 2014). It raises an interesting question that why the weight of pupal body is significantly increased in the BmFib-H gene knock-out Bombyx mori line (FGKO).…”

Advanced technologies for genetically manipulating the silkwormBombyx mori, a model Lepidopteran insect

“…Rearing those individuals to adults and backcrossing them to wild-type individuals resulted in 0.28% of the larval progeny in which BmBLOS2 was targeted having the oily phenotype [66]. More recently, fibroin heavy chain gene (BmfibH), encoding the largest and most abundant silk protein, was successfully disrupted using ZFNs [68]. These examples, while demonstrating the functionality of ZFNs in B. mori, also showed that obtaining heritable germline mutations using this technology was inefficient and complex-it is generally a difficult and time-consuming task to genetically modify genes using ZFNs owing to the challenge of designing and building ZFs with sufficient specificity, which are mainly restricted by the lack of known ZFs for some trinucleotide sequences and context effects of individual ZFs in an array [69,70].…”