Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

Shota Nakade, Takuya Tsubota, Yuto Sakane, Satoshi Kume, Naoaki Sakamoto, Masanobu Obara, Takaaki Daimon, Hideki Sezutsu, Takashi Yamamoto, Tetsushi Sakuma () and Ken-ichi T. Suzuki ()
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Shota Nakade: Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
Takuya Tsubota: Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan
Yuto Sakane: Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
Satoshi Kume: Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
Naoaki Sakamoto: Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
Masanobu Obara: Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
Takaaki Daimon: Insect Growth Regulation Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan
Hideki Sezutsu: Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan
Takashi Yamamoto: Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
Tetsushi Sakuma: Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
Ken-ichi T. Suzuki: Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan

Nature Communications, 2014, vol. 5, issue 1, 1-8

Abstract: Abstract Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.

Date: 2014
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DOI: 10.1038/ncomms6560

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