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Chinese Journal of Biotechnology ; (12): 4342-4350, 2021.
Article in Chinese | WPRIM | ID: wpr-921510


The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.

Animals , Bombyx/metabolism , CRISPR-Cas Systems/genetics , Endonucleases/genetics , Gene Editing , RNA, Guide/genetics
Chinese Journal of Biotechnology ; (12): 100-108, 2020.
Article in Chinese | WPRIM | ID: wpr-787684


Bombyx mori is a lepidopteran insect with important economic value. Bombyx mori nucleopolyhedrovirus (BmNPV) causes huge economic loss to silkworm industry in China every year. The objective of this study is to determine the anti-BmNPV mechanism of Bombyx mori strain NC99R, and to provide a basis for understanding the molecular mechanism of the silkworm resistance strain. The normal control Dazao (DZ) strain and the NC99R resistant strain were fed with occlusion bodies (OB). The median lethal dose (LD50) analysis of the DZ and NC99R showed that the LD50 of DZ was 1.2×10⁵ OBs/larva, while NC99R was 1.8×10⁶ OBs/larva. The LD50 of the NC99R was about 15 times higher than the DZ. The mortality of DZ and NC99R were analyzed, which were fed with 1×10⁶ OBs/larva and injection with 1×10⁶ BVs/larva. The results showed that the death peak of DZ was concentrated in the 4th to 6th day. And the death peak of NC99R was concentrated in the 6th to 8th day, with a delay of 1-2 days compared with the control. The BmNPV DNA copy number showed that the BmNPV genome in DZ proliferated rapidly. The copy number of BmNPV DNA in NC99R were increased slowly after oral infection and body injection. HE staining showed that midgut tissue has no significant difference between DZ and NC99R in the early stage of oral infection. At 96 h p.i., the nucleus of DZ midgut became larger and shedding. The NC99R had enlarged nuclei, but the cells were still arranged neatly. Finally, the expression of virus genes in different periods were analyzed by RT-PCR. The results indicated that the immediate early gene ie-1 expression levels began to down-regulate after 24 h p.i.. The early, late, and extremely late genes were also down-regulated, and finally maintained at a lower expression level.