Non-coding CRISPR interference screen to identify regulatory elements of Xist in TX1072 XX SP107 mESCs at the onset of random X inactivation

Developmental genes are controlled by complex cis-regulatory landscapes that integrate multiple signals to ensure the correct spatio-temporal expression pattern. To investigate the underlying regulatory principles, we use the Xist locus as a model, which encodes the master regulator of X-chromosome inactivation. Xist is upregulated at the primed pluripotent state in a female-specific manner, thus integrating developmental cues and X-dosage information. It remains poorly understood how these signals are decoded by the ~800kb genomic region that controls Xist. While a series of repressive cis-regulatory elements have been identified, the distal enhancers that activate Xist transcription remain largely unknown. Here we use an inducible CRISPRi system in a non-coding CRISPR screen to profile 138 candidate regulatory elements on their effect on Xist upregulation during early differentiation of mouse Embryonic Stem Cells (mESCs). Overall design: A non-coding CRISPRi screen for Xist was performed in differentiating mESCs (TX1072 XX SP107) carrying an ABA-inducible dCas9-KRAB system. The library contained 7058 gDNAs targeting 138 candidate regulatory elements within the X inactivation center, as well as 300 non-targeting controls. After 2 days of differentiation via 2i/LIF-withdrawal, the cells were stained for Xist RNA using a FlowFISH protocol and sorted into 4 different fractions (Negative, Low, Medium and High) corresponding to 15% bins of Xist-expression.