Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Graduate Program in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Science. 2024 Mar 22;383(6689):1344-1349. doi: 10.1126/science.adj3566. Epub 2024 Mar 21.
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.
大型 DNA 组装方法是合成原核生物和芽殖酵母染色体的里程碑式成就的基础。虽然芽殖酵母通过约 125 个碱基对 DNA 序列定义的着丝粒来控制染色体的遗传，但哺乳动物和许多其他真核生物使用大型的、表观遗传的着丝粒。利用着丝粒表观遗传学可以形成人类人工染色体（HAC），但不足以避免最初的 DNA 分子在引入细胞时发生猖獗的多聚化。我们描述了一种有效形成单拷贝 HAC 的方法。它使用一个约 750 千碱基对的构建体，这个大小足以容纳存在于内着丝粒和外着丝粒的不同染色质类型，从而避免了多聚化的需要。通过利用酵母原生质体融合来简化向哺乳动物细胞的传递。这些发展使得在后生动物细胞的背景下进行忠实的染色体工程成为可能。
