TALEN-mediated genome editing of the ku80 gene in the silkworm Bombyx mori

Abstract

Genome editing is a powerful tool for the functional analysis of targeted genes. We have previously found that a novel knock-in system, precise integration into target chromosome (PITCh), allows the integration of a donor vector harboring the hsp90 promoter and GFP into the silkworm biogenesis of lysosome-related organelles complex 1, subunit 2 gene in a precise and efficient manner. Here, we examined whether this technique can be used for the knock-in of other silkworm genes. The ku80 gene was selected as a target and was efficiently mutagenized using a pair of transcription activator-like effector nucleases (TALENs) that were constructed for its specific cleavage. Knock-in was then carried out using these TALENs. Microinjection of TALEN mRNAs mixed with the donor vector resulted in significant expression of the GFP marker in G0 larvae. Importantly, GFP expression was also detected in G1 individuals, suggesting that the integrated donor vector can be inherited in the next generation. Genotyping analysis showed that the donor vector was inserted into the targeted ku80 locus in four individuals. It was further found to be inherited in the G2 generation in a Mendelian manner. We argue that the PITCh system offers a versatile tool for the knock-in of various genes, and should contribute to the further promotion of sericultural studies.