Bombyx mori kynurenine 3‐monooxygenase gene editing and insect molecular breeding using the clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 system
Autor: | Kee Young Kim, Jeong Won Hong, Seong-Wan Kim, Jeong Hee Yu, Sang Kuk Kang, Nam-Suk Kim, Chan Young Jeong, Jong Woo Park, Su-Bae Kim |
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Rok vydání: | 2020 |
Předmět: |
Gene Editing
0106 biological sciences Genetics Molecular breeding biology Cas9 fungi 010401 analytical chemistry DNA Shuffling Gene mutation Bombyx biology.organism_classification 01 natural sciences Genome 0104 chemical sciences Kynurenine 3-Monooxygenase Genome editing Bombyx mori 010608 biotechnology Animals CRISPR CRISPR-Cas Systems Gene Biotechnology |
Zdroj: | Biotechnology Progress. 36 |
ISSN: | 1520-6033 8756-7938 |
Popis: | Genome editing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9, a third-generation gene scissors, and molecular breeding at the genome level are attracting considerable attention as future breeding techniques. In the present study, genetic and phenotypic analyses were conducted to examine the molecular breeding of Bombyx mori through CRISPR/Cas9-mediated editing of the kynurenine 3-monooxygenase (KMO) gene. The synthesized guide RNAs (gRNAs) were analyzed using T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA, and Cas9 complexes were microinjected into silkworm embryos. After microinjection, the hatching rate and the incidence of mutation were determined as 18.1% and 60%, respectively. Gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed; however, certain embryos and moths produced through sib-mating had significant differences compared to the wild-type. In successive generations, a distinct phenotypic change was also observed by continuous mating. Thus, although there are limitations in the phenotypic expression in breeding through the induction of deletion mutations, as in the present study, the process is believed to yield successful results within a shorter period compared to traditional breeding and is safer than transgenic technology. |
Databáze: | OpenAIRE |
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