title: Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.
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.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Nakade
authors.affiliation: Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Tsubota
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors.forename: Yuto
authors.initials: Y
authors.lastname: Sakane
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kume
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Sakamoto
authors.affiliation: Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Obara
authors.affiliation: Insect Growth Regulation Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Daimon
authors.affiliation: Transgenic Silkworm Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Sezutsu
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors.forename: Ken-ichi T
authors.initials: KT
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D001483:Base Sequence; D012831:Bombyx; D064113:CRISPR-Cas Systems; D004247:DNA / Q000378:metabolism*; D003851:Deoxyribonucleases; D055879:Gene Knock-In Techniques / Q000379:methods*; D005818:Genetic Engineering; D005822:Genetic Vectors; D059765:Homologous Recombination; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D012441:Saccharomyces cerevisiae; D014981:Xenopus
chemical_list: D004247:DNA; D003851:Deoxyribonucleases