Genetic Engineering Of Bombyx Silk Gland Using Genome Editing

Evolution is one of the most powerful things in the world. It has produced some remarkable creatures, of which silk gland is a fascinating organ that exists in a variety of insects and almost half of the34,000spider species. The most notable feature of silk gland is the impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to25%) without aggregation or denaturalization2. This ability was greatly enhanced in silkworm, Bombyx mori, by thousands of years’domestication and breeding. Eating about20g mulberry leaves, one commercial B. mori larvae can produce0.5g pure silk protein (dry weight,25%of total worms). With the developing understanding of disease pathogens and the identification of new molecular targets, biopharmaceutical proteins such as vaccines, hormones and protein-based biomaterials are in increasing demand for both analytical and clinical applications. Bioreactors using genetic modified organisms, which enable production of recombinant proteins in a commercial scale, emerged and underwent decades of development to meet this demand. The impressive ability to secrete and store huge amount of pure silk protein, together with the potential to be large scale production, make the silk gland of B. 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. Since the first protein (human collagen by Tomita, et al. in2003) was produced in the silk gland of B. mori, several encomoically important proteins including FGF, interferon, HAS and antibodies were subsequently expressed. But none of these proteins went into the commercial production stage, mainly due to the extemenly low production yield and difficulties to purify thses proteins from transgenic cocoons. We used to think a lot about these two problems. Why the silk gland is able to produce huge amount of silk proteins, but not other proteins? This is also might be a consequence of perfect evolution. During the evolution and domestication of silk gland, all the mechanism and pathway might have been adapted to serve the silk protein production. Meanwhile, the silk proteins will undergo a self assemble process to form silk, which is very troubling for the protein purification. Thus we proposed that reducing or removal of the endogenous silk proteins might increase the expression level of exogenous recombinant proteins. To verifiy this hypothesis, we first showed that knocking down of BmSerl by transgenic RNAi could increase the expression of foreign DsRed protein in MSG. Then we established a highly efficient genome editing platform using ZFN, TALEN and CRISPR/Cas9to knock out silk protein coding genes. BmFib-H mutated silk gland was demonstrated to be an ideal bioreactor for the production of recombinant proteins and protein-based materials, the production yield of which can reach up to50%. Furthermore, we also obtained the BmFib-L mutant and established a method to simultaneously knock out multiple silk coding genes. The major achievments and conclusions are as following:1. Transgenic knock down of BmSerl could increase the level of DsRedWe proposed a novel strategy to increase the expression yield of foreign proteins in silk gland of B. mori. A transgenic vector designed to knock down the expression of BmSerl gene through transgenic RNAi was constructed. Microinjection of the vector into embyos of Dazao strain generated14transgenic lines, and the mRNA level of BmSerl in1of the4tested lines was knocked down by50%. Then,4transgenic lines that express DsRed in the MSG were generated, and1line was selected. Fluorescent detection revealed that the excessive expression of DsRed in MSG will affect the silk spinning process. When two selected lines were crossed, we found that the expression of DsRed was enhanced in both mRNA and proteins level, when BmSerl was knocked down. These results suggested that the strategy we proposed here worked as expected.2. A highly efficient genome editing platform was establishedIn recent years, the emergence and rapid application of site-specific genome editing tools such as ZFN, TALEN and CRISPR/Cas9has greatly reshaped the field of genetic manipulation. We established genome editing platforms in B. mori at the very first time when a new tool was invented. We also optimized genome editing from impossible to possible, then to highly efficient, and finally to efficient and simple in B. mori. During this process, several interesting results were obtained:(1) An efficient assembly system of the customized TALEN B. mori genome editing using a modified Golden Gate ligation strategy was constructed.(2) An evaluation system for binding activity and cleavage efficiency of TALEN using a luciferase (LUC) reporter system in insect cell culture was established.(3) The ability of TALEN to induce both mosaic and heritable mutations were demonstrated.(4) TALEN was also showed to induce targeted genomic structural variation of an8.9mb segment.(5) The ability of TALEN to induce heritable large chromosomal deletion.(6) The CRISPR/Cas9system for B. mori genome editing was established and optimized.(7) A method for precise genomic strucrtural variation was established.(8) CRISPR/Cas9was showed to mediate targeted mutagenesis and genomic structural variation.(9) A strategy and procedure to perform heritable genome editing of target genes with no significant phenotype effect was showed.(10) The mechanism of chromosomal duplication was demostrrated.3. BmFib-H mutants were generated and its new functions were revealedWe performed targeted mutagenesis of fibroin heavy chain (BmFib-H) gene, which encodes the largest and most abundant silk protein, using custom ZFN. And the genotyping and phenotyping results showed that:(1) BmFib-H mutants contain two different types of mutant sequences, one is in-frame and the other one if frame shift.(2) BmFib-H could generate both dominnat and recessive mutats.(3) The mechanism of dominant mutation is different from known mechanisms.(4) The PSG of BmFib-H mutant was smaller, with a normal cells number but smaller and abnormal cells, while ASG and MSG were as normal.(5) The lumen of BmFib-H mutants PSG was hollow inside, and MSG was almost empty with a very small amount of sericin proteins secreted by MSG cells.(6) The silk filaments within the BmFib-H mutant cocoons were much thinner and exhibited a sericin-cocoon like phenotype, and contained only sericins and some small fibroin proteins.(7) BmFib-H mutant cocoons were much lighter and thinner, while the pupae were little heavier, indicating a nutrient resorption from cocoon to pupae.(8) The abnormal phenotype of PSG cells may provide some important information to reveal the regulation of PSG cell size increase.(9) The detection of both Fib-L and P25proteins in the cocoons of BmFib-H mutant suggested that the H-L subunit is only necessary for the intracellular transport, secretion, and luminal transport of large fibroin proteins such as BmFib-H. The aforementioned results not only provided new evidence for the traditional functions of BmFib-H gene, but also opened some interesting new questions. 4. The empty silk gland is suitable for highly efficient recombinant protein production.To determine whether BmFib-H mutant can be used as a host for recombinant protein expression, we first attempted to express a recombinant enhanced green fluorescent protein (EGFP). One transgenic B. mori line was generated from the BmFib-H mutant, by piggyBac mediated transformation. Fluorescence could be detected in the PSG cells and lumen of the whole silk gland. The Gel electrophoresis and Western blotting analysis of the cocoons showed a significantly increase in the expression level of recombinant fusion protein. The estimated content was50%of total cocoons. This high productivity is superior to any of the known transgenic bioreactors (the highest was15%in the case of EGFP fusion protein production that we reported previously) and cultured cell factories (the highest was27g/L in the case of IgG production using an IgG-producing PER.C6clone29). Therefore, the genome edited silk gland generated here shows great promise as a highly efficient system for large scale production of EGFP fusion protein and perhaps other recombinant proteins.5. The first synthetic silk fiber was produced in the empty silk glandGiven its "empty" feature and "full" protein production ability, we suspected that Fib-H-I1silk gland could be an ideal system for expression of protein based materials. For this demonstration, we designed an artificial silk protein (art-Fib-H) by assembling N terminal (contains the1st repetitive region and1st amorphous domain) and C terminal (contains the11th amorphous domain and12th repetitive region) regions of BmFib-H. For easy detection, an EGFP protein was fused between1st and11th amorphous domains. One transgenic B. mori line, Fib-H-I1-TR, in which art-Fib-H was expressed under the control of a BmFib-H promoter, was obtained. Fluorescence detection showed that art-Fib-H could be expressed in PSG cells and secreted into silk gland lumen. The cocoons of Fib-H-I1-TR were thicker and1.7fold heavier, and the cocoon shell rate was also higher. SEM analysis of the cocoons revealed that silk fibers of Fib-H-I1-TR were thicker than Fib-H-I1and formed typical bifilar fibers in the inner layer of cocoon, which is the typical phenotype of fibroin containing fibers in the inner layer. The expression of art-Fib-H was also confirmed by gel analysis and western blotting. The mechanical properties of cocoons were also analyzed and the result showed that the cocoons of Fib-H-I1-TR were much stronger than Fib-H-I1, indicating art-Fib-H proteins were correctly assembled into silk fibers. The successful production of artificial silk designed from BmFib-H protein has showed the possibility to utilize Fib-H-I1silk gland as a unique high efficient bioreactor for the production of protein-based materials and will surely pave the way for production of other biomaterials.6. BmFib-L mutants were generated and mothod to generate multiple silk coding genes knock out was establishedTo achieve higher expression yield and more "empty" silk gland bioreactor, we also proposed to knock out the other silk coding genes. First, we performed the knock out of BmFib-L and obtained its mutants. As there are more than six silk coding genes, it will take too long to knock out them one by one. Therefore, we established a method to knock out multiple genes simultaneously. This method was first approved by one-step manipulating6genes in BmNs cell lines. Then we demonstrated the possibility to knock out BmP25, BmSerl and BmPSer3in both cell lines and embryos.In conclusion, the hypothesis, methods and major findings of in this thesis are unprecedented in both fields of silkworm and bioreactors. Knocking down/out the endogenous silk protein coding genes to increase the expression of foreign proteins, this strategy not only overcomed the two major bottlenecks of silk gland bioreactors, sweeping the obstacles of comercialization of silk gland bioreactor, but also praved a new way for the establishment of other bioreactors. Genome editing is the most revolutionized genetic manipulation tool in modern genetics. Aiming at knocking out silk coding genes, we have established a highly efficient genome editing platforms in B. mori, and performed some chanllaging, cutting edge investigations about genome editing using B. mori as a model system. Genome editing really means too much for B. mori, and our results not only realized this long-lasting dream in the field of B. mori reseach, but also provide more possibilities for functinal genomics studies in this magic species. Beside the innovations on strategy and technologies, the "empty silk gland" generated here will be a new start and new hope for the development of silk gland bioreactor.