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Precise genome editing in the silkworm Bombyx mori using TALENs and ds- and ssDNA donors - A practical approach
Y. Takasu, I. Kobayashi, T. Tamura, K. Uchino, H. Sezutsu, M. Zurovec,
Jazyk angličtina Země Anglie, Velká Británie
Typ dokumentu časopisecké články
- MeSH
- bourec genetika MeSH
- DNA genetika metabolismus MeSH
- editace genu metody MeSH
- jednovláknová DNA genetika metabolismus MeSH
- TALENs genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Engineered nucleases are able to introduce double stranded breaks at desired genomic locations. The breaks can be repaired by an error-prone non-homologous end joining (NHEJ) mechanism, or the repair process can be exploited to introduce precise DNA modifications by homology-directed repair (HDR) when provided with a suitable donor template. We designed a series of DNA donors including long dsDNA plasmids as well as short ssDNA oligonucleotides and compared the effectiveness of their utilization during gene targeting with highly efficient transcription activator-like effector nucleases (TALENs). While the use of long dsDNA donors for the incorporation of larger DNA fragments in Bombyx is still a problem, short single-stranded oligodeoxynucleotides (ssODNs) are incorporated quite efficiently. We show that appropriately designed ssODNs were integrated into germ cells in up to 79% of microinjected individuals and describe in more detail the conditions for the precise genome editing of Bombyx genes. We specify the donor sequence requirements that affected knock-in efficiency, and demonstrate the successful applications of this method of sequence deletion, insertion and replacement in the Bombyx genome.
Citace poskytuje Crossref.org
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