RRC ID 56907
Author Takasu Y, Kobayashi I, Tamura T, Uchino K, Sezutsu H, Zurovec M.
Title Precise genome editing in the silkworm Bombyx mori using TALENs and ds- and ssDNA donors - A practical approach.
Journal Insect Biochem. Mol. Biol.
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.
Volume 78
Pages 29-38
Published 2016-11
DOI 10.1016/j.ibmb.2016.08.006
PII S0965-1748(16)30124-2
PMID 27569417
MeSH Animals Bombyx / genetics* DNA / genetics* DNA / metabolism DNA, Single-Stranded / genetics* DNA, Single-Stranded / metabolism Gene Editing / methods* Transcription Activator-Like Effector Nucleases / genetics* Transcription Activator-Like Effector Nucleases / metabolism
IF 3.562
Resource
Silkworms p50T w1-pnd