Versatile NHEJ-based Knock-in using genome editing (VIKING) method

The VIKING method is a genome editing method that uses the more frequently occurring non-homologous end joining (NHEJ) repair pathway instead of the conventional homologous recombination (HR) repair pathway in the genome editing method for cultured cells. The advantage is that an insertion of a large vector exceeding 10kbp is available, which is difficult with a viral vector. Moreover, a sequence that serves as a marker such as a drug resistance gene is inserted into the genome sequence, which facilitates a cell selection after genome editing. We also succeeded in dramatically reducing unintended sequence modification (random integration) by optimizing the introduction ratio of three vectors. In case that the target is the coding region of the gene, an insertion of a donor vector can establish a gene-disrupted clone, and it has been used in various cell types.

Genome-edited cultured cell production by VIKING method

Reference

1. Hashimoto, M. and Takemoto, T*. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci. Rep. 5, 11315; doi: 10.1038/srep11315 (2015).
2. Hashimoto M, Yamashita Y, Takemoto T*.Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev. Biol. 418: 1-9 (2016).
3. Tanihara F, Takemoto T*, Kitagawa E, Rao S, Do L, Onishi A, Yamashita Y, Kosugi C, Suzuki H, Sembon S, Suzuki S, Nakai M, Hashimoto M, Yasue A, Matsuhisa M, Noji N, Fujimura T, Fuchimoto Di, Otoi T*. Somatic cell reprogramming-free generation of genetically modified pigs. Science Advances. 2 (9) e1600803 (2016).