Background

Traditional chromosome substitution strains (CSS) have been invaluable for studying complex traits, but they face significant limitations. Conventional methods rely on iterative backcrossing and marker-assisted selection, making them time-consuming and prone to unintended recombination. More critically, these approaches are restricted to intra-species systems and cannot accommodate chromosomes from different species or synthetic sources due to reproductive and centromere compatibility issues. The TEAM platform was developed to overcome these constraints and enable targeted chromosome replacement in mammalian embryonic stem cells (ESCs).

Methodology: The TEAM Platform

The TEAM platform integrates two key technologies. First, CRISPR/Cas9 is used to eliminate target chromosomes from recipient cells. Second, MMCT transfers intact donor chromosomes—whether natural or engineered—into the recipient ESCs. For proof-of-concept, researchers focused on Y chromosome replacement, performing both intra-species substitutions (mouse-to-mouse) and cross-species transfers (human-to-mouse). The transferred chromosomes were tagged with GFP markers to enable tracking. Resulting ESCs were injected into tetraploid blastocysts to generate live animals, allowing comprehensive assessment of chromosome stability, developmental outcomes, and molecular consequences through whole-genome sequencing, FISH analysis, and transcriptomic profiling.

Key Findings: Stability and Compatibility

Intra-species Y chromosome replacement proved highly successful. Mouse-to-mouse substitutions yielded ESCs with stable karyotypes and normal gene expression, and the resulting mice developed normally into adulthood with maintained chromosome integrity. However, cross-species replacement revealed severe complications. Human Y chromosomes exhibited poor stability in mouse ESCs, with rapid GFP signal loss, significant gene deletions, and progressive DNA damage across passages. Mice carrying human Y chromosomes suffered high neonatal mortality, growth retardation, and systemic inflammation. Critically, the study identified centromere incompatibility as the root cause: human Y chromosome centromeres showed dramatically reduced CENP-A protein levels, leading to chromosome missegregation, micronucleus formation, and extensive chromosomal rearrangements.

Significance

This research establishes TEAM as a powerful, modular tool for chromosome biology research and synthetic karyotype design. More importantly, it demonstrates that centromere compatibility is the critical determinant of cross-species chromosome stability. The findings reveal how centromere dysfunction drives chromosome instability and inflammatory disease phenotypes, highlighting fundamental barriers to xenochromosomal engineering. Future applications may extend beyond Y chromosomes to autosomes and synthetic chromosomes, provided that centromere compatibility challenges can be addressed through artificial centromere engineering or chimeric CENP-A rescue strategies.
DOI:10.1093/procel/pwag010