Protein-only centromeric chromatin assembly streamlines human artificial chromosome formation

Area of Science:

• Molecular Biology
• Genetics
• Epigenetics

Background:

• Human artificial chromosomes (HACs) are crucial for studying chromosomal elements and delivering large genetic payloads.
• Previous limitations included the inability to create large HACs (multiple Mb) and the requirement for genetically engineered recipient cells for epigenetic centromere seeding.

Purpose of the Study:

• To design and construct a significantly larger HAC (2 Mb) than previously reported.
• To develop a robust, cell-engineering-free method for epigenetic centromere seeding and HAC formation.

Main Methods:

• Construction and delivery of a 2 Mb HAC DNA construct to human cells.
• Utilized a novel protein-based system for immediate, cytoplasm-initiated epigenetic centromere seeding.
• Assessed HAC inheritance in recipient cells without selection pressure.

Main Results:

• Successfully created and delivered a 2 Mb HAC, approximately three times larger than prior generations.
• The new epigenetic seeding method functions immediately upon delivery, bypassing the need for recipient cell genetic modification.
• HACs were faithfully inherited across cell divisions without the need for selective markers.

Conclusions:

• This study reports the first generation of large (2 Mb) HACs.
• A highly efficient, selection-free HAC formation method was established, applicable to unmanipulated human cells.
• Functional centromere formation within the same cell cycle as HAC delivery is key to high-efficiency HAC generation.