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MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems

Nature Protocols volume 11, pages 118133 (2016) | Download Citation

Abstract

Programmable nucleases enable engineering of the genome by utilizing endogenous DNA double-strand break (DSB) repair pathways. Although homologous recombination (HR)-mediated gene knock-in is well established, it cannot necessarily be applied in every cell type and organism because of variable HR frequencies. We recently reported an alternative method of gene knock-in, named the PITCh (Precise Integration into Target Chromosome) system, assisted by microhomology-mediated end-joining (MMEJ). MMEJ harnesses independent machinery from HR, and it requires an extremely short homologous sequence (5–25 bp) for DSB repair, resulting in precise gene knock-in with a more easily constructed donor vector. Here we describe a streamlined protocol for PITCh knock-in, including the design and construction of the PITCh vectors, and their delivery to either human cell lines by transfection or to frog embryos by microinjection. The construction of the PITCh vectors requires only a few days, and the entire process takes 1.5 months to establish knocked-in cells or 1 week from injection to early genotyping in frog embryos.

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Acknowledgements

The authors express their appreciation to A. Kawahara and Y. Hisano (University of Yamanashi, Yamanashi, Japan) for co-developing the modified PITCh system. We also thank H. Ochiai (Hiroshima University, Hiroshima, Japan) for sharing the synthesized mNeonGreen cDNA under the license agreement with Allele Biotechnology and Pharmaceuticals, Inc. This work was supported by the Japan Society for the Promotion of Science (25890014 to T.S., 25124708 to K.-I.T.S. and 26290070 to T.Y.), the Sasakawa Foundation (to S.N.), the Uehara Memorial Foundation (to T.S.) and the Ministry of Health, Labor, and Welfare of Japan (to T.Y.).

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Affiliations

  1. Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan.

    • Tetsushi Sakuma
    • , Shota Nakade
    • , Yuto Sakane
    • , Ken-Ichi T Suzuki
    •  & Takashi Yamamoto

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Contributions

T.S. organized and wrote the manuscript. S.N. performed the human cell experiments and wrote the manuscript concerning human cell procedures. Y.S. performed the frog experiments. K.-I.T.S. wrote the manuscript concerning frog procedures. T.Y. supervised the work.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Tetsushi Sakuma.

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DOI

https://doi.org/10.1038/nprot.2015.140

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