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セミナー 学会・シンポジウム 市民公開講座
第37回生命医科学セミナー【京都大学 Dr. Knut Woltjen】
- [開催場所]総合研究棟1階 102講義室
演 題：Endogenous DNA repair pathways for gene editing in human induced pluripotent stem cells.
演 者： Dr. Knut Woltjen
Hakubi Center for Advanced Research, Dept. of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University
場 所：病院地区総合研究棟1階 102講義室
Combining induced pluripotent stem cell (iPSC) and nuclease-mediated genome engineering tools such as CRISPR/Cas9 holds great promise to study human genetic diseases. Programmable nucleases induce DNA double strand breaks (DSBs) which recruit endogenous DNA repair machinery to the target locus. Error-prone repair of DSBs, often attributed to non-homologous end joining (NHEJ), is commonly used to produce gene knockouts. Interestingly, we have found that NHEJ is often precise, and that many deletions can be attributed to microhomology-mediated end joining (MMEJ). In fact, we can employ MMEJ to generate disease-relevant micro-deletions in otherwise normal iPSCs.
Template-mediated homology-directed repair (HDR) has long been used to create designer gene modifications. Our lab uses HDR to introduce reporter transgenes into safe-harbour loci for tracking iPSCs during differentiation and in mice following transplantation. In the interest of recapitulating disease-causing mutations, we have developed a novel protocol to engineer single point mutations in human iPSCs. Our method uses conventional gene targeting by HDR followed by seamless excision of antibiotic resistance cassettes guided by engineered microhomology and endogenous MMEJ machinery. The efficiency and fidelity of our method is demonstrated by the creation human iPSCs which elicit disease-relevant metabolic phenotypes affecting purine salvage pathways. The strategies discussed may be generally applied to other mammalian cell lines and disease models.
問合せ：九州大学大学院 医学研究院 基礎放射線医学分野 續 輝久（内線6141）