Rapid assembly of functional modules for generating human artificial chromosome constructs compatible with epigenetic
Gabriel J Birchak1,2,3,4, Daniel G Gibson5, Praveen Kumar Allu1,2,3
1Department of Biochemistry & Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104 USA.
Biorxiv : the Preprint Server for Biology
|November 19, 2025
Summary
Researchers developed a new method for building human artificial chromosomes (HACs) using bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). This efficient technique rapidly assembles complex genetic modules for advanced gene delivery and research.
Area of Science:
- Genetics and Molecular Biology
- Synthetic Biology
- Chromosomal Engineering
Background:
- Human artificial chromosomes (HACs) are crucial for studying centromere function and delivering large genetic payloads.
- Previous HAC construction methods, particularly those using yeast artificial chromosomes (YACs), faced challenges with multimerization and complex assembly.
- Transformation-associated recombination (TAR) strategies for YAC construction can be cumbersome when incorporating multiple functional modules.
Purpose of the Study:
- To develop a more efficient and streamlined approach for assembling complex multi-module HAC constructs.
- To overcome the limitations of traditional TAR strategies for large-scale YAC-based HAC engineering.
- To facilitate the rapid testing and incorporation of diverse genetic modules and cargoes into HACs.
Main Methods:
- Modules were assembled in vitro using high-fidelity methods within a bacterial artificial chromosome (BAC) format.
- Assembled BAC modules were transferred into a recipient YAC using a simplified TAR strategy.
- Rapid validation of clones was achieved through whole-genome Oxford Nanopore Technologies (ONT) sequencing and de novo assembly.
Main Results:
- The new approach demonstrated high efficiency, with two-thirds of screened yeast clones containing the correct TAR product.
- Whole-genome ONT sequencing and assembly successfully validated the designed multi-module YAC constructs.
- The verified multi-module HACs were efficiently fused to human cells, forming functional HACs upon seeding with CENP-A nucleosomes.
Conclusions:
- This BAC-to-YAC assembly strategy significantly simplifies and accelerates the engineering of complex HACs.
- The method allows for rapid incorporation of various functional modules and genetic cargoes, enabling tailored HAC design.
- This advance holds promise for future applications in gene therapy, disease modeling, and fundamental genetic research.
