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bioRxiv · 10.1101/2025.10.02.680129

Rapid assembly of functional modules for generating human artificial chromosome constructs compatible with epigenetic centromere seeding

Birchak, G. J.·Gibson, D. G.·Allu, P. K.·Kashyap, P.·Glass, J. I.·Black, B. E.
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Abstract

The ongoing development of human artificial chromosomes (HACs) will permit investigation into essential centromere processes and the means to deliver large genetic cargoes to target cells. Starting with large ([~]750 kb) yeast artificial chromosome (YAC)-based constructs limits the rampant multimerization that has complicated many prior types of HACs. Large YAC construction is accomplished using transformation-associated recombination (TAR) strategies that can become unwieldly when several functional modules are to be incorporated and tested. To address this issue, we developed an approach where modules are built using high-fidelity in vitro assembly strategies in a bacterial artificial chromosome (BAC) format. Then, the assembled modules are transferred in a simplified TAR step into a recipient YAC harboring the prokaryotic "stuffer" DNA that comprises a large portion of the final HAC construct. This approach is highly efficient with two-thirds of all screened yeast clones harboring the correct TAR product. Further, whole-genome Oxford Nanopore Technologies (ONT) sequencing/alignments, de novo assembly of the final YAC using a single ONT sequencing run, and close inspection of highly repetitive regions are all streamlined to rapidly validate clones that match the design. The fully sequenced, verified strain harboring a multi-module construct was then fused to human cells, where it efficiently formed functional HACs upon initial seeding with CENP-A-containing nucleosomes. We envision that the rapid assembly steps will be useful to quickly incorporate different functional modules, including diverse genetic cargoes, to engineer HACs with specific design features.

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Birchak, G. J., Gibson, D. G., Allu, P. K., Kashyap, P., Glass, J. I., Black, B. E.. 2025-10-03. Rapid assembly of functional modules for generating human artificial chromosome constructs compatible with epigenetic centromere seeding. https://doi.org/10.1101/2025.10.02.680129

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