Hashizume Ryotaro, Wakita Sachiko, Sawada Hirofumi, Takebayashi Shin-Ichiro, Kitabatake Yasuji, Miyagawa Yoshitaka, Hirokawa Yoshifumi S, Imai Hiroshi, Kurahashi Hiroki
Department of Pathology and Matrix Biology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan.
Department of Genomic Medicine, Mie University Hospital, Tsu, Mie 514-8507, Japan.
PNAS Nexus. 2025 Feb 18;4(2):pgaf022. doi: 10.1093/pnasnexus/pgaf022. eCollection 2025 Feb.
Human trisomy 21, responsible for Down syndrome, is the most prevalent genetic cause of cognitive impairment and remains a key focus for prenatal and preimplantation diagnosis. However, research directed toward eliminating supernumerary chromosomes from trisomic cells is limited. The present study demonstrates that allele-specific multiple chromosome cleavage by clustered regularly interspaced palindromic repeats Cas9 can achieve trisomy rescue by eliminating the target chromosome from human trisomy 21 induced pluripotent stem cells and fibroblasts. Unlike previously reported allele-nonspecific strategies, we have developed a comprehensive allele-specific (AS) Cas9 target sequence extraction method that efficiently removes the target chromosome. The temporary knockdown of DNA damage response genes increases the chromosome loss rate, while chromosomal rescue reversibly restores gene signatures and ameliorates cellular phenotypes. Additionally, this strategy proves effective in differentiated, nondividing cells. We anticipate that an AS approach will lay the groundwork for more sophisticated medical interventions targeting trisomy 21.
导致唐氏综合征的人类21三体是认知障碍最常见的遗传病因，仍是产前诊断和植入前诊断的关键焦点。然而，旨在从三体细胞中消除多余染色体的研究有限。本研究表明，通过成簇规律间隔短回文重复序列Cas9进行等位基因特异性多染色体切割，可通过从人类21三体诱导多能干细胞和成纤维细胞中消除目标染色体来实现三体拯救。与先前报道的等位基因非特异性策略不同，我们开发了一种全面的等位基因特异性（AS）Cas9靶序列提取方法，可有效去除目标染色体。DNA损伤反应基因的临时敲低会增加染色体丢失率，而染色体拯救可逆转地恢复基因特征并改善细胞表型。此外，该策略在分化的非分裂细胞中也被证明有效。我们预计，等位基因特异性方法将为针对21三体的更复杂医学干预奠定基础。
