Efficient formation of single-copy human artificial chromosomes

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Ha...

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Bibliographic Details
Published in:Science (American Association for the Advancement of Science) 2024-03, Vol.383 (6689), p.1344-1349
Main Authors: 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
Format: Article
Language:English
Subjects:
Animals
Artificial chromosomes
Centromere - genetics
Chromatin
Chromosomes
Chromosomes, Artificial, Human - genetics
DNA
Eukaryotes
Genomes
Human artificial chromosomes
Humans
Mammals
Mammals - genetics
Saccharomyces cerevisiae - genetics
Saccharomycetales - genetics
Yeasts
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Summary:Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~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 ~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.
ISSN:0036-8075
1095-9203
DOI:10.1126/science.adj3566