CRISPR screens decode dual regulatory logic at the Xist locus

The team from the Max Planck Institute for Molecular Genetics in Berlin began with a pooled CRISPR interference screen targeting 570 transcription factor genes in differentiating female mouse embryonic stem cells. Using a split dCas9–KRAB system that represses gene expression without cutting DNA, the team transduced cells with guide RNA libraries and sorted populations by Xist RNA levels detected through fluorescence in situ hybridisation. This screen identified 24 previously unknown Xist activators and 15 repressors, including the surprising discovery that OCT4 – previously classified as a repressor – acts as the most potent activator during differentiation, alongside the X-linked factor ZIC3 and formative pluripotency regulator OTX2.

To connect transcription factors to specific regulatory elements, the team developed an innovative CRISPR screening variant using fluorescent reporters. They generated cell lines carrying individual Xist regulatory elements upstream of a minimal promoter driving GFP, then performed pooled CRISPR interference screens on 12 reporter lines. Sorting cells by GFP intensity revealed 166 functional transcription factor–regulatory element interactions, uncovering distinct modules. Proximal elements near the Xist promoter responded to transiently upregulated factors, such as ZIC3, which peak before Xist activation. On the contrary, distal enhancers within Jpx, Ftx, and Xert were controlled by later-acting factors, including OTX2.

Integration with time-course RNA sequencing revealed that factors controlling proximal elements showed XX-biased expression, suggesting they govern female-specific binary activation decisions. In contrast, factors targeting distal elements responded primarily to developmental signals and function to boost Xist RNA levels independently of sex. The functional importance emerged through single-cell RNA sequencing: the deletion of distal elements reduced Xist levels approximately twofold and significantly impaired chromosome-wide gene silencing, with dose–response analysis showing that silencing efficiency increases gradually with Xist expression.

The work was led by Till Schwämmle, Edda Schulz and colleagues from the Max Planck Institute for Molecular Genetics in Berlin. It was published in Nature Structural & Molecular Biology today 6 October 2025.