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

Area of Science:

• Genetics
• Molecular Biology
• Synthetic Biology

Background:

• Human artificial chromosomes (HACs) are crucial for studying centromere function and delivering large genetic payloads.
• Previous HAC development faced challenges like multimerization and complex assembly using yeast artificial chromosomes (YACs).

Purpose of the Study:

• To develop a more efficient and streamlined method for constructing multi-module HACs.
• To facilitate the rapid assembly and validation of HACs for diverse applications.

Main Methods:

• Utilized high-fidelity in vitro assembly in bacterial artificial chromosomes (BACs) for module construction.
• Employed a simplified transformation-associated recombination (TAR) step to transfer BAC modules into a YAC.
• Leveraged Oxford Nanopore Technologies (ONT) sequencing for rapid clone validation and de novo assembly.

Main Results:

• Achieved high efficiency, with two-thirds of screened yeast clones containing the correct TAR product.
• Successfully constructed and validated a multi-module HAC using the developed approach.
• Demonstrated the formation of functional HACs in human cells post-fusion.

Conclusions:

• The novel BAC-to-YAC assembly strategy significantly enhances the efficiency of multi-module HAC construction.
• This method enables rapid engineering of HACs with specific design features and diverse genetic cargoes.
• The streamlined validation process accelerates the development of HACs for genetic research and therapeutic applications.