ゲノム編集技術の登場によりゲノム上の遺伝子のノックアウトは比較的容易に達成できるようになったが, ノックインは未だ困難である。本研究では, 薬剤選抜を利用した標的組換えベクターの簡便構築方法を考案, TALEN発現ベクターおよびCRISPR発現ベクターと共導入することで, 様々な疾患患者由来のiPS細胞で原因遺伝子の修復や, 正常なiPS細胞に変異導入に成功した。特にCRISPRについては, 複数個所をオフターゲット切断のリスク低く切断可能なall-in-oneベクターの簡便構築法を考案し, これと2種の薬剤選抜可能な標的組換えベクターを共導入することで, 確実にホモノックイン株を樹立可能な系を完成させた。
Genome editing technology has made it familiar for us to knock-out a gene, but it is still difficult to knock-in a certain DNA sequence precisely into a genomic locus. In this study, a simple and effective method was developed to construct a gene knock-in vector with a drug-resistant gene cassette. Using the vector in combination with TALEN of CRISPR expression vectors, responsible genes of iPSC lines derived from various disease patients were successfully corrected or those of control iPSC lines were modified to have mutation. Especially for CRISPR expression vector, an all-in-one construct was developed to express two sets of sgRNAs for two dual-nickase Cas9 pairs to cut two genomic loci at the same time for lower the risk of off-target effect. Combination of this vector with two knock-in vectors with two different drug-resistant gene cassette, a reliable homo knock-in system was also developed and confirmed for its feasibility.
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