VIKING : How it works
Welcome to the VIKING method
Easy and fast knock-in for any cell line!
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The VIKING (Versatile non-homologous end joining-based knock-in module for genome editing) method includes two tips to improve knock-in using the NHEJ repair pathway.
Tip 1: Use an optimal ratio of the genome editing vectors for transfection (75% of selected clones harbor knock-ins without random integrations).
Tip 2: By using the VKG1 sequence, almost all vectors harboring a pUC backbone can be used as knock-in donor vectors WITHOUT any modification.
pUC, pBluescript, pcDNA, pENTR ... various standard vectors can be used as knock-in donor vectors.
"Development of versatile non-homologous end joining-based knock-in module for genome editing"
Shun Sawatsubashi, Yudai Joko, Seiji Fukumoto, Toshio Matsumoto, Shigeo S. Sugano
Scientific Reports, 2018
"Protocol for CRISPR/Cas9-based knock-in using the VIKING method"
Yudai Joko, Shigeo S. Sugano, Seiji Fukumoto, Toshio Matsumoto, Shun Sawatsubashi
Protocol Exchange, 2019
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Team
Shun Sawatsubashi (Tokushima University)
Shigeo S. Sugano (National Institute of Advanced Industrial Science and Technology)
Working principle
The VIKING method utilizes a non-homologous end joining (NHEJ) repair-based knock-in technique such as ObLiGaRe1-2. ObLiGaRe achieves a highly efficient knock-in because it does not harness homologous recombination (HR) but instead uses NHEJ repair. This is a dominant DNA repair pathway that contrasts with the HR repair pathway3. NHEJ-based knock-in occurs more frequently than HR-based knock-in1-2, and involves linearization of the donor vector inside the cells. Therefore, three circular plasmids, a donor vector, a donor-cleavage vector, and a genome-cleavage vector, should be simultaneously transfected. Various reports showed successful knock-in using CRISPR/Cas9 and NHEJ-based knock-in strategies4-10. The VIKING method uses optimal conditions for NHEJ-based knock-in, taking into account random integrations. It also uses the VKG1 sequence, which exists in the backbones of various vectors, for donor cleavage to save reconstruction of both the donor-cleavage vector and the donor vector.
Validated cell lines (at January 2018):
HaCaT (human, aneuploid keratinocyte cell line)
HEK293F (human, embryonic kidney cells)
C4-2 (human, prostate cancer cell line)
UMR-106 (rat, osteogenic cell line)
Conflict of interest
Patents relating to the VIKING method were filed by Tokushima University.
If you are interested in using the patent of the VIKING method, please contact shun-sawa2@tokushima-u.ac.jp
The VIKING method can be used freely for academic research.
Related studies:
1. Maresca et al. Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res 23, 539-546 (2013).
2. Cristea et al. In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration. Biotechnol Bioeng 110, 871-880 (2013).
3. Haber "Genome stability" 2013. Garland Science
4. Auer et al. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res 24, 142-153 (2014).
5. Lackner et al. A generic strategy for CRISPR-Cas9-mediated gene tagging. Nat Commun 6, 10237 (2015).
6. Kimura et al. Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering. Sci Rep 4, 6545 (2014).
7. Schmid-Burgk et al. CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism. Nat Commun 7, 12338 (2016).
8. Li et al. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nat Plants 2, 16139 (2016).
9. Katoh et al. Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system. Mol Biol Cell 28, 538 898–906 (2017).
10. Tálas et al. A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid. DNA Res 24, 609–621 (2017).