Efficient formation of single-copy human artificial chromosomes

• 成果类型: Article
• 署名作者: Gambogi, Craig W.; Birchak, Gabriel J.; Mer, Elie; Brown, David M.; Yankson, George; Kixmoeller, Kathryn; Gavade, Janardan N.; Espinoza, Josh L.; Kashyap, Prakriti; Dupont, Chris L.; Logsdon, Glennis A.; Heun, Patrick; Glass, John I.; Black, Ben E.
• 署名单位: University of Pennsylvania; University of Pennsylvania; University of Pennsylvania; University of Pennsylvania; University of Pennsylvania; J. Craig Venter Institute; University of Edinburgh; Technical University of Darmstadt; University of Pennsylvania
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-11644
• DOI: 10.1126/science.adj3566
• 发表日期: 2024-03-22
• 页码: 1344-1349
• 关键词: aurora b; dna; centromere; chromatin; yeast; hac; CONSTRUCTION; cloning; ORGANIZATION; methylation

摘要

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through similar to 125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. We describe an approach that efficiently forms single-copy HACs. It employs a similar to 750-kilobase construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.