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.

Biology Centre of the ASCR Branisovska 31 370 05 Ceske Budejovice Czech Republic

Institute of Agrobiological Sciences National Agriculture and Food Research Organization 1 2 Owashi Tsukuba Ibaraki 305 8634 Japan

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The Exact Timing of Microinjection of Parthenogenetic Silkworm Embryos Is Crucial for Their Successful Transgenesis

Functional Analysis of Adipokinetic Hormone Signaling in Bombyx mori