Protein-only centromeric chromatin assembly streamlines human artificial chromosome formation
Human artificial chromosomes (HACs) are inherited through cell divisions alongside natural chromosomes, serving as tools for interrogating chromosomal elements and as vectors for large genetic cargoes (1-5). Despite recent progress (6-8), large (i.e., multiple Mb) HACs have not been reported. Further, HAC formation via epigenetic seeding of centromeric chromatin currently requires prior engineering of recipient human cells (6-8), hampering their potential deployment in many useful cell types. Here, we designed, built, and delivered to human cells a 2 Mb HAC construct that is [~]3 times larger than the prior generation. We also report a robust epigenetic centromere seeding approach that initiates immediately upon delivery to the human cell cytoplasm and bypasses genetic engineering of target cells. The HACs are then faithfully inherited in the absence of selection. Thus, formation of functional centromeric chromatin in the same cell cycle of HAC delivery drives high efficiency HAC formation. TeaserA system for efficient protein-only centromere seeding permits visualizing the first step of HAC formation