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KMID : 0613820190290050537
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Journal of Life Science
2019 Volume.29 No. 5 p.537 ~ p.544
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Biogenesis of Lysosome-related Organelle Mutant Silkworms by Direct Injection of a Cas9 Protein-guided RNA Complex into Bombyx mori Embryos
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Kim Kee-Young
Yu Jeong-Hee
Kim Su-Bae
Kim Seong-Wan
Kim Seong-Ryul
Choi Kwang-Ho
Kim Jong-Gil
Park Jong-Woo
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Abstract
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Genome editing technology employing gene scissors has generated interest in molecular breeding in various fields, and the development of the third-generation gene scissors of the clustered, regularly interspaced short palindromic repeat (CRISPR) system has accelerated the field of molecular breeding through genome editing. In this study, we analyzed the possibility of silkworm molecular breeding using gene scissors by genomic and phenotypic analysis after editing the biogenesis of lysosome-related organelles (BmBLOS) gene of Bakokjam using the CRISPR/Cas9 system. Three types of guide RNAs (gRNA) were synthesized based on the BmBLOS gene sequence of Bakokjam. Complexes of the prepared gRNA and Cas9 protein were formed and introduced into Bombyx mori BM-N cells by electroporation. Analysis of the gene editing efficiency by T7 endonuclease I analysis revealed that the B4N gRNA showed the best efficiency. The silkworm genome was edited by microinjecting the Cas9/B4N gRNA complex into silkworm early embryos and raising the silkworms after hatching. The hatching rate was as low as 18%, but the incidence of mutation was over 40%. In addition, phenotypic changes were observed in about 70% of the G0 generation silkworms. Sequence analysis showed that the BmBLOS gene appeared to be a heterozygote carrying the wild-type and mutation in most individuals, and the genotype of the BmBLOS gene was also different in all individuals. These results suggest that although the possibility of silkworm molecular breeding using the CRISPR/Cas9 system would be very high, continued research on breeding and screening methods will be necessary to improve gene editing efficiency and to obtain homozygotes.
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KEYWORD
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BmBLOS, Bombyx mori, CRISPR/Cas9, gene editing, guide RNA
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