Journal
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY
Volume 78, Issue -, Pages 29-38Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.ibmb.2016.08.006
Keywords
Engineered nucleases; HDR; DSB; Oligonucleotide donors; Genotyping; Homologous recombination
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Funding
- JSPS KAKENHI [23580083]
- Grant Agency of the Academy of Sciences of Czech Republic [P305/14-27816S]
- Grants-in-Aid for Scientific Research [23580083] Funding Source: KAKEN
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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. (C) 2016 Elsevier Ltd. All rights reserved.
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