Genome editing of marsupial RSX reveals conserved and divergent principles in mammalian X-inactivation

Courtois, A.; Menchero, S.; Ogushi, S.; Varsally, W.; Wood, S.; Decarpentrie, F.; Yoshimi, R.; Shiraishi, A.; Inoue, K.; Abe, T.; VandeBerg, J. L.; Snell, D. M.; Kiyonari, H.; Turner, J. M. A.

doi:10.64898/2026.07.01.735830

bioRxiv · 10.64898/2026.07.01.735830

Genome editing of marsupial RSX reveals conserved and divergent principles in mammalian X-inactivation

Courtois, A.·Menchero, S.·Ogushi, S.·Varsally, W.·Wood, S.·Decarpentrie, F.·Yoshimi, R.·Shiraishi, A.·Inoue, K.·Abe, T.·VandeBerg, J. L.·Snell, D. M.·Kiyonari, H.·Turner, J. M. A.

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Abstract

X-chromosome inactivation balances X-gene dosage between the sexes in eutherians and marsupials. The lncRNA Xist mediates X-inactivation in eutherians but is absent from marsupials. An evolutionarily unrelated lncRNA, RSX, has been identified in marsupials, but its role in X-inactivation is unresolved because genome editing in these mammals has only recently become feasible. Using CRISPR-Cas9 in the opossum, we show that RSX is required for the initiation of X-inactivation in marsupial embryos. RSX deletion later, in differentiated cells, causes modest X-gene de-repression, with repressive chromatin marks H3K27me3 and H3K9me3 retained. H2AK119ub is not enriched on the opossum inactive X, revealing divergent Polycomb-mediated regulation in therians. Our findings reveal stage-specific roles of RSX in marsupial X-inactivation and identify conserved and divergent features of X-dosage compensation in mammals.

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Courtois, A., Menchero, S., Ogushi, S., Varsally, W., Wood, S., Decarpentrie, F., Yoshimi, R., Shiraishi, A., Inoue, K., Abe, T., VandeBerg, J. L., Snell, D. M., Kiyonari, H., Turner, J. M. A.. 2026-07-07. Genome editing of marsupial RSX reveals conserved and divergent principles in mammalian X-inactivation. https://doi.org/10.64898/2026.07.01.735830

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