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Nat Commun. 2014 Nov 20;5:5560. doi: 10.1038/ncomms6560.

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

Author information

1
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
2
Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
3
Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
4
Insect Growth Regulation Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.

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.

PMID:
25410609
PMCID:
PMC4263139
DOI:
10.1038/ncomms6560
[Indexed for MEDLINE]
Free PMC Article

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