Application Of CRISPR/Cas9 System In Silkworm Gene Function Studies

The silkworm Bombyx mori is an important insect of spinning cocoons and also one of model organisms for biological research. CRISPR/Cas9 system from one adaptive immunity system of bacteria was developed in recent two years. After artificial modification by researchers, this system has been become a flourishing genome editing technology and used in many model organisms. In this study, the genes correlated with cuticle color formation and silk gland development in silkworm were selected respectively. These genes were destroyed by CRISPR/Cas9 system, and mutants were obtained. Finally, we successfully established an efficient and rapid genome knockout technology in silkworm, and it will be one powerful tool for silkworm functional genomics research. In the meantime, the silkworm of lose-of-function in silk gland gene was analyzed by RNA-seq to elucidate the molecular mechanisms of silk gland development. The main results are as follows:1. We successfully destroyed 4 different genes (Bm-ok、BmKMO、BmTHvBmtan) about silkworm cuticle color and transparency. According to the target design principle,8 different targets were selected (3 for BmTH,4 for both Bm-ok and Bmtan, and 1 for BmKMO) within exons. High quality Cas9-mRNA and sgRNA were synthesized in vitro and mixed together and injected into the newly eggs. The results showed that all the 8 targets worked and the mutation frequencies were about 16.7%-35.0%. But different targets for different genes had diverse frequencies, for instance, only 16.7% of mutation frequenc was observed for two targets of Bmtan. Because of the randomness of Cas9 cutting, their specific mutations induced by the CRISPR/Cas9 system presented various types. Their indel numbers were unpredictable. When several sgRNA for one gene were injected into embryos, maybe only 1 target worked or all targets worked simultaneously, which resulted in large fragment deletion. But only 1 target working also can make large fragment deletion, like BmKMO, deletions as long as 240 bp and 289 bp were found at its target location. Generally, off-targeting is a potential issue about the application of Cas9/sgRNA technology in genome editing. However, in our test, we did not observe any obvious off-targeting effects, indicating that the application of the CRISPR/Cas9 technique could have high specificity. In this study, we obtained the mutant of Bm-ok with mosaic phenotype of Go and translucent epidermis of G1 homozygote, like "oily silkworm".2. We successfully destroyed the Bmsage gene, which was considered as the homologous gene of salivary gland-expressed bHLH in Drosophila using CRISPR/Cas9. One specific target was designed on the basis of Bmsage structure. And its mutation frequency was 46.7% that was more efficient than other 4 genes in cuticle characters. Randomly,7 most similar sites were selected to estimate its off-targeting effect and also no obvious off-targeting effect happened. Bmsage mutant was screened by sequencing and the phenotype from G2 offspring different from WT. In the screened homozygous mutants, silk glands developed abnormally and only the whole ASG and a small part of PSG remained with PSG absent. The mature mutant larvae were incapable of spinning and making cocoons. The survival mutants became naked pupae finally, but could emerge into moths, mate and lay eggs. Some of mutants died during pupation with keeping the larvae form or abnormal pupae. In addition, we dissected 1st molting larvae (1st instar larvae) and found they had one semblable phenotype of silk gland of 5th instar mutant. Two different kinds of indel mutations from offspring of Go mosaic silkworms were separated and exhibited the same abnormal silk gland structure without MSG and PSG. This illustrated that the Bmsage mutant phenotype was due to Bmsage knockout. So Bmsage maybe participates in embryonic generation and development of the silk gland.3. Two kinds of crossing strategies were used for Go mosaic in our study. Through Crossing Strategy 1 (mosaic x WT), we estimated the germline transmission frequency of mutations. A 28.6% frequency of germline mutation transmission was observed for Bm-ok and even 75% for Bmsage. So this CRISPR/Cas9 system has high efficiency of mutation transmission from Go to Gi, which is enough for screen homozygote from offspring. Crossing Strategy 2 (mosaic x mosaic) is a method to get homozygous mutants quickly and shorten the screen time. For Bm-ok the frequency of homozygote reached above 90% with the complete translucent phenotype using Crossing Strategy 2. However, these homozygotes may not be genuine because possibly the two alleles possess different sequences, which resulted in the compound heterozygous.4. RNA-seq was performed for Bmsage mutant missing silk gland. We preliminarily got the knowledge of physiological and biochemical processes which Bmsage maybe participate in. GO analysis indicated that lose-of-function in Bmsage mainly affected some cellular components in differentially expressed genes. For instance, the myosin and troponin genes associated with muscle function showed obvious up-regulation, and most of lectin and glycosidase genes associated with carbohydrate metabolic process also showed obvious up-regulation except some with down-regulation. Moreover, in the top 10 GO terms, most of differentially expressed genes were chitinase genes associated with chitinase activity and several serine protease inhibitor genes. KEGG data showed that Bmsage was mostly related to pathways of carbohydrate, lipid and amino acid metabolism. Among them most were carbohydrate metabolism, such as galactose, glycosaminoglycan, amino sugar and nucleotide sugar metabolism. So we conclude that Bmsage is possibly involved in many carbohydrate metabolisms. In these differentially expressed genes, chitinase, beta-N-acetylglucosaminidase and serine protease inhibitor genes maybe are the most relevant to silk gland formation and development in silkworm. Moreover some tRNA genes are also related to the silk gland formation, such as TRNAE-CUC, TRNAN-GUU, TRNAD-GUC and TRNAV-GAC. Therefore, Bmsage perhaps involved in the differentiation and formation of silk gland through a complex regulatory network.