Novel Approaches of (Neo)Centromere Seeding on Human Chromosomes

Centromeres are chromosomal loci that are central to the fidelity of chromosome segregation during cell division. Importantly, human centromeres are also characterised by tandem arrays of AT-rich alpha-satellite (alphoid) DNA repeats. However, centromeric alphoid DNA is not essential to nucleate a functional centromere. This is evident from the discovery of spontaneously formed centromeres at naïve loci, for instance in human patients. These neocentromeres epitomize the epigenetic nature of centromeres, nucleating at sites devoid of alphoid DNA. Indeed, centromeres are epigenetically defined by the presence of the histone H3 variant CENP-A at centromeric chromatin. Significantly, CENP-A can induce de novo centromere formation independent of DNA sequence. Taking advantage of this, centromeres offer a unique opportunity to exploit their epigenetic nature to develop robust artificial centromeres (and subsequently chromosomes) for synthetic biology/gene therapy applications. This proposal will take advantage of the latest developments in CRISPR-Cas9 and long-read sequencing technologies to develop inducible methods of (neo)centromere seeding that bypass the need for repetitive alphoid DNA sequences (Aim 1 and 2). We will then take advantage of these strategies to answer fundamental questions of centromere competition and inactivation in human cells (Aim 3).