High fidelity human chromosome transfer and elimination

The ability to synthesize human and other Gb-scale genomes requires new technologies. Operating on human chromsomes or chromosome scaffolds - by iterative recombination of synthetic DNA contained in microbial episomes - will be facilitated by their isolation in mESC 'assembly cell'. However, methods reported for chromosome transfer commonly lead to large-scale DNA damage, and existing methods for chromosome elimination have primarily been demonstrated in cancer cells. Methods for high fidelity chromosome transfer and elimination in non-transformed cells have remained elusive. Here, we directly transfect isolated human chromosomes into mESCs to generate monochromosomal hybrids, and transfer human chromosomes from these hybrids to human cells by rapid (R)-MMCT to generate defined synthetic trisomies. We effect the net replacement of an endogenous human chromosome with the corresponding transferred human chromosome to generate diploid human cells. While we observe the telomeres of human chromosomes lengthen to the set point of mouse telomeres in mESCs and return to the set point of human cells post-elimination, we show that the chromosome transfers and eliminations proceed in non-transformed cells with minimal genetic alteration.