A programmable platform enabling targeted chromosome substitution and cross-species stability profiling.

Area of Science:

• Mammalian genetics
• Chromosome engineering
• Synthetic biology

Background:

• Chromosome substitution strains (CSS) are valuable for dissecting complex traits.
• Conventional CSS development is limited by iterative breeding and intraspecific compatibility.
• Novel methods are needed for efficient and programmable chromosome replacement.

Purpose of the Study:

• To develop a novel platform for creating chromosome substitution strains.
• To enable programmable chromosome replacement using CRISPR/Cas9 and microcell-mediated chromosome transfer (MMCT).
• To investigate the stability and functional consequences of substituting mouse Y chromosomes with homologous or heterologous Y chromosomes.

Main Methods:

• Developed the Targeted chromosome Elimination And Microcell-mediated chromosome transfer (TEAM) platform.
• Combined CRISPR/Cas9-mediated chromosome elimination with MMCT for chromosome replacement.
• Generated intraspecies (mouse Y) and interspecies (human Y) chromosome substitutions in mice.

Main Results:

• Intraspecies Y chromosome substitutions resulted in stable embryonic stem cells and viable adult males.
• Interspecies human Y chromosome substitutions led to severe instability, DNA damage, and inflammation.
• Human Y chromosome instability was linked to reduced CENP-A levels, leading to segregation errors and rearrangements.

Conclusions:

• The TEAM platform facilitates programmable construction of mammalian chromosome substitution models.
• Interspecies chromosome substitution poses significant stability challenges, impacting animal health and development.
• This technology offers new avenues for investigating chromosomal function, genome evolution, and synthetic karyotype design.