Aquarius depletion leads to R-loop related replication stress and discoordination of transcription and replication, leading to cell identity maintenance perturbation

Embryonic stem cells (ESCs) combine rapid proliferation with the a capacity to differentiate into diverse lineages. Their high replication origin density and short G1-phase raise the risk of transcription–replication conflicts (TRCs), which threaten genome stability. Here, we show that mouse ESCs (mESCs) display exceptionally poor coordination between transcription and replication. Remarkably, this causes only a modest increase in TRCs, which can be explained by redundant expression of the majority of known TRC resolution factors leading to high TRC turnover rates. A targeted siRNA screen against TRC factors revealed the RNA:DNA helicase Aquarius as a key factor protecting the high genomic traffic of mESCs. Aquarius depletion disrupts transcription–replication coordination, induces replication stress, DNA damage, and increases cell state heterogeneity. Thus, Aquarius is critical for preserving the pluripotent identity of mESCs.