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

The functional integrity of a mammalian chromosome depends on its long-term co-evolution with species-specific nuclear environment. How chromosomes co-adapt with their native environment to define 3D architecture and transcription remains unclear, largely due to a lack of experimental models that dissect this co-evolution. Here, we report cross-species chromosome substitution (CROSS), a method enabling stable, scarless replacement of host chromosomes with divergent orthologs. We integrate microcell-mediated chromosome transfer with CRISPR/Cas9 to import the intact 152-Mb rat X chromosome into male mouse embryonic stem cells, and subsequently replace the mouse X chromosome, maintaining its stability and integrity. Rat-specific LINE1 and RatSatRep2 repeats recruit mouse SETDB1 inefficiently, causing local H3K9me3 loss. This triggers 3D remodeling, characterized by de novo topologically associating domain (TAD) boundaries that aberrantly activate adjacent genes, including Rhox5, impairing differentiation. We provide a platform for dissecting sequence and epigenome cooperation in chromosome regulation, and evaluating fidelity of large-scale heterologous DNA. Chromosome function depends on coevolution with nuclear environment, but how is unclear. Here, authors use CROSS to stably and scarlessly replace mouse chrX with rat chrX. Rat repeats poorly recruit SETDB1, causing H3K9me3 loss, new TAD boundaries, that dysregulate genes, impairing differentiation.