講演情報
[P6-1]CRISPR/Cas9を介した染色体転座による迅速なヒト染色体クローニング
○宮本 人丸1, 山崎 匡太郎1, 宇野 愛海2, 阿部 智3, 濱道 修生3, 岸間 菜々美1, 冨塚 一磨2, 香月 康宏1,3 (1.鳥取大学 大学院 医学系研究科 医科学専攻 染色体医工学分野, 2.東京薬科大学 生命科学部 応用生命科学科 生物工学研究室, 3.鳥取大学 染色体工学研究センター)
We have generated trans-chromosomic (Tc)-animal models of human congenital diseases by using artificial chromosomes to investigate disease mechanisms, including mouse models of Down syndrome containing human chromosome 21 (34 Mb) and Duchenne muscular dystrophy containing human mutated dystrophin gene (2.4 Mb). Since these Mb-sized genes and chromosome loci are too large to be loaded onto plasmids or BACs, mouse artificial chromosomes (MACs) have been used to generate Tc-mice in addition to the advantages of stable maintenance and germline transmission. However, loading human chromosome loci onto the MACs requires more than seven steps of cloning and screening, which is time- and effort-consuming. Specifically, 1) generation of hybrid cells of the human-mouse A9 cell line, which is resistant to mitotic inhibitors and capable of chromosome transfer, and 2) introduction of chromosome into DT40 cells with high homologous recombination efficiency to insert the loxP sequence have been major technological limitations. Here we report the successful transfer of a single human chromosome 6 directly from human iPS cells into CHO cells. Furthermore, we are attempting to develop a strategy wherein translocation of the 6p21.3 HLA gene cluster into the MACs is achieved by genome editing with CRISPR/Cas9. This method bypasses conventional chromosome modification methods using human A9 hybrids and DT40 cells. This method allows rapid human chromosome cloning into the MACs in three steps and can be adapted to new research targets and diseases.