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KMID : 0613820200300080701
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Journal of Life Science
2020 Volume.30 No. 8 p.701 ~ p.707
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Artificial Mutation for Silkworm Molecular Breeding Using Gene Scissors
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Hong Jeong-Won
Jeong Chan-Young
Yu Jeong-Hee
Kim Su-Bae
Kang Sang-Kuk
Kim Seong-Wan
Kim Nam-Suk
Kim Kee-Young
Park Jong-Woo
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Abstract
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Gene editing technology using the clustered regularly interspaced short palindromic repeat (CRISPR) and the CRISPR associated protein (Cas)9 has been highly anticipated in developing breeding techniques. In this study, we discuss gene scissors as a tool for silkworm molecular breeding through analysis of Bombyx mori Kynurenine 3-Monooxygenase (BmKMO) gene editing using the CRISPR/Cas9 system and analysis of generational transmission through mutagenesis and selective crossing. The nucleotide sequence of the BmKMO gene was analyzed, and three guide RNAs (gRNAs) were prepared. Each synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA and Cas9 complexes were subsequently microinjected into the silkworm embryos; the hatching rate was 18% and the incidence of mutation was 60%. The gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed. In homozygotes generated by self-crossing, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/ Cas9 system is possible and could be an effective way of shortening the time required.
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KEYWORD
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Bombyx mori, BmKMO, CRISPR/cas9, gene editing, gRNA
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