A Programmable Platform Enabling Targeted Chromosome Substitution and Cross-Species Stability Profiling

Chromosome substitution strains (CSS) are critical tools for dissecting complex traits, although iterative breeding steps and intraspecific compatibility requirements limit conventional approaches. Here, we developed an optimized TEAM platform for chromosome replacement combing CRISPR/Cas9–mediated chromosome elimination with microcell-mediated chromosome transfer (MMCT). Using this approach, we substituted the endogenous mouse Y chromosome (chrY) with either the mouse or human Y chromosome. Intraspecies substitutions yielded karyotypically stable embryonic stem cells that supported development into adult males. By contrast, in interspecies CSS, human chrY displayed severe instability and progressive DNA damage. Despite partial transcription of human chrY genes, recipient animals exhibited systemic inflammation, high rates of neonatal death, and poor growth. Reduced CENP-A levels were observed at human chrY centromeres, leading to segregation errors, micronuclei formation, and widespread chromosome rearrangements. This technology enables programmable construction of chromosome substitution models for investigating chromosomal function, genome evolution, and synthetic karyotype design in mammals.