title: Precise genome editing in the silkworm Bombyx mori using TALENs and ds- and ssDNA donors - A practical approach.
abstract: 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.
authors: Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors: Yoko
authors: Y
authors: Takasu
authors: Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors: Isao
authors: I
authors: Kobayashi
authors: Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors: Toshiki
authors: T
authors: Tamura
authors: Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors: Keiro
authors: K
authors: Uchino
authors: Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors: Hideki
authors: H
authors: Sezutsu
authors: Biology Centre of the ASCR, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic.
authors: Michal
authors: M
authors: Zurovec
mesh_terms: D000818:Animals; D012831:Bombyx / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics* / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D004247:DNA; D000069896:Transcription Activator-Like Effector Nucleases
keywords: DSB; Engineered nucleases; Genotyping; HDR; Homologous recombination; Oligonucleotide donors