CROSS: A cross-species chromosome substitution platform for dissecting chromosome architecture and activity

The functional integrity of a mammalian chromosome is shaped by its long-term, co-evolution with species-specific nuclear environment. How chromosomes co-adapt with their native environment to define 3D architecture and transcriptional activity remain poorly understood, largely due to a lack of experimental models capable of systematically dissecting this co-evolution relationship. Here, we report a cross-species chromosome substitution (CROSS) method, a robust genomic engineering method that enables the stable, scarless replacement of host chromosomes with evolutionarily divergent orthologs. By integrating microcell-mediated chromosome transfer with CRISPR/Cas9, we imported the intact 158-Mb rat X chromosome into mouse embryonic stem cells, and subsequently achieved targeted substitution of its endogenous mouse counterpart, maintaining stability and integrity. Using this model, we found that rat-specific LINE1 and RatSatRep2 repeats failed to adequately recruit host SETDB1 in mouse cells, leading to localized erosion of H3K9me3 heterochromatin. This further triggered 3D structural remodeling, characterized by the de novo formation of topologically associating domain (TAD) boundaries that aberrantly activated adjacent genes—including Rhox5 , the master regulator of the Rhox cluster—impairing cellular differentiation. Our method provides a powerful chromosome engineering platform for dissecting how genomic sequences and epigenetic mechanisms cooperate in regulating chromosome architecture and function, and for evaluating the structural and functional fidelity of large-scale synthetic or heterologous DNA across species.