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タイトル: | Microhomology-assisted scarless genome editing in human iPSCs |
著者: | Kim, Shin-Il Matsumoto, Tomoko Kagawa, Harunobu Nakamura, Michiko Hirohata, Ryoko Ueno, Ayano Ohishi, Maki Sakuma, Tetsushi https://orcid.org/0000-0003-0396-1563 (unconfirmed) Soga, Tomoyoshi Yamamoto, Takashi Woltjen, Knut https://orcid.org/0000-0003-2293-1183 (unconfirmed) |
著者名の別形: | 曽我, 朋義 山本, 卓 |
キーワード: | CRISPR-Cas9 genome editing Genetic engineering Stem-cell biotechnology |
発行日: | 5-Mar-2018 |
出版者: | Springer Nature |
誌名: | Nature Communications |
巻: | 9 |
論文番号: | 939 |
抄録: | Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes. |
記述: | ゲノム編集技術を用いたヒトiPS細胞での正確な一塩基置換技術(MhAX法)を開発. 京都大学プレスリリース. 2018-03-06. |
著作権等: | © The Author(s) 2018. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
URI: | http://hdl.handle.net/2433/229515 |
DOI(出版社版): | 10.1038/s41467-018-03044-y |
PubMed ID: | 29507284 |
関連リンク: | https://www.kyoto-u.ac.jp/ja/research-news/2018-03-06-0 |
出現コレクション: | 学術雑誌掲載論文等 |
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