講演情報
[O28-1]CRISPR/Cas9システムの染色体削減と核型修正への応用
○橋詰 令太郎1, 脇田 幸子1, 原 万里1, 澤田 博文2, 竹林 慎一郎3, 北畠 康司4, 宮川 世志幸5, 倉橋 浩樹6 (1.三重大学 医学系研究科 修復再生病理学, 2.三重大学 大学院医学系研究科 集中治療学, 3.三重大学大学院 生物資源学研究科, 4.大阪大学 大学院医学系研究科 小児科学, 5.日本医科大学 大学院医学系研究科 分子遺伝学, 6.藤田医科大学 総合医科学研究所 分子遺伝学研究部門)
Introduction Down syndrome (DS) is a genetic disorder with a known genetic cause of a triplication of the human chromosome 21 (HSA21) and is the most common survivable aneuploidy. However, little research has been conducted to address how we eliminate excess chromosomes. Herein, we constructed a Cas9 system that allele-specific cleaves targeted chromosomes at multiple locations, and examined the efficiency of excess chromosome removal in trisomy 21 induced pluripotent stem (iPS) cells.
Methods Trisomy 21 iPS cells were electroporated with SpCas9 and sgRNA(s) co-expression plasmid vectors, selected by temporary drug pressure followed by downstream analyses including FISH with chromosome 21 specific probes and G-banding, or short tandem repeat analysis after single cell cloning.
Results The analysis of FISH revealed that haplotype-aware multiple chromosome cleavages induce chromosome loss in a cleavage site number-dependent manner. We found that all the lost chromosomes are the target chromosomes of the CRISPR/Cas9 system. Furthermore, inhibition of non-homologous end joining by knockdown of POLQ and LIG4 increased the efficiency of chromosome elimination. The G2-M cell cycle phase elongation decreased the efficacy indicating chromosome elimination as a cell cycle-dependent phenomenon. The efficiency of karyotype correction was higher with the allele-specific chromosome-breaking method than with the allele-nonspecific method.
Discussion Chromosome breaks by CRISPR/Cas9 are useful as a karyotype correction method for aneuploidy with excess chromosomes.
Methods Trisomy 21 iPS cells were electroporated with SpCas9 and sgRNA(s) co-expression plasmid vectors, selected by temporary drug pressure followed by downstream analyses including FISH with chromosome 21 specific probes and G-banding, or short tandem repeat analysis after single cell cloning.
Results The analysis of FISH revealed that haplotype-aware multiple chromosome cleavages induce chromosome loss in a cleavage site number-dependent manner. We found that all the lost chromosomes are the target chromosomes of the CRISPR/Cas9 system. Furthermore, inhibition of non-homologous end joining by knockdown of POLQ and LIG4 increased the efficiency of chromosome elimination. The G2-M cell cycle phase elongation decreased the efficacy indicating chromosome elimination as a cell cycle-dependent phenomenon. The efficiency of karyotype correction was higher with the allele-specific chromosome-breaking method than with the allele-nonspecific method.
Discussion Chromosome breaks by CRISPR/Cas9 are useful as a karyotype correction method for aneuploidy with excess chromosomes.