Dissecting molecular phenotypes through FACS-based pooled CRISPR screens

Pooled CRISPR screens are emerging as a powerful tool to dissect regulatory networks, by assessing how a protein responds to genetic perturbations in a highly multiplexed manner. A large number of genes are perturbed in a cell population through genomic integration of one single-guide RNA (sgRNA) per cell. A subset of cells with the phenotype of interest can then be enriched through fluorescence-activated cell sorting (FACS). SgRNAs with altered abundance after phenotypic enrichment allow identification of genes that either promote or attenuate the investigated phenotype. Here we provide detailed guidelines on how to design and execute a pooled CRISPR screen to investigate molecular phenotypes. We describe how to generate a custom sgRNA library and how to perform a FACS-based screen using readouts such as intracellular antibody staining or Flow-FISH to assess phosphorylation levels or RNA abundance. Through the variety of available perturbation systems and readout options many different molecular and cellular phenotypes can now be tackled with pooled CRISPR screens

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of a distal enhancer element

To ensure dosage compensation for X-linked genes between the sexes, one X chromosome is silenced during early embryonic development of female mammals. This process of X-chromosome inactivation (XCI) is initiated through upregulation of the RNA Xist from one X chromosome shortly after fertilization. Xist then mediates chromosome-wide gene silencing in cis and remains expressed in all cell types except the germ line and the pluripotent state, where XCI is reversed. The factors that drive Xist upregulation and thereby initiate XCI remain however unknown. We identify GATA transcription factors as potent Xist activators and demonstrate that they are essential for the activation of Xist in mice following fertilization. Through a pooled CRISPR activation screen we find that GATA1 can drive ectopic Xist expression in murine embryonic stem cells (mESCs). We demonstrate that all GATA factors can activate Xist directly via a GATA-responsive regulatory element (RE79) positioned 100 kb upstream of the Xist promoter. Additionally, GATA factors are essential for the induction of XCI in mouse preimplantation embryos, as simultaneous deletion of three members of the GATA family (GATA1/4/6) in mouse zygotes effectively prevents Xist upregulation. Thus, initiation of XCI and possibly its maintenance in distinct lineages of the preimplantation embryo is ensured by the combined activity of different GATA family members, and the absence of GATA factors in the pluripotent state likely contributes to X reactivation. We thus describe a form of regulation in which the combined action of numerous tissue-specific factors can achieve near-ubiquitous expression of a target gene

Distal and proximal cis-regulatory elements sense X chromosome dosage and developmental state at the Xist locus.

Developmental genes such as Xist, which initiates X chromosome inactivation, are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatiotemporal expression patterns. Xist integrates information on X chromosome dosage and developmental stage to trigger X inactivation in the epiblast specifically in female embryos. Through a pooled CRISPR screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist at the onset of random X inactivation. Chromatin profiling reveals that X-dosage controls the promoter-proximal region, while differentiation cues activate several distal enhancers. The strongest distal element lies in an enhancer cluster associated with a previously unannotated Xist-enhancing regulatory transcript, which we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. With this study, we start to disentangle how multiple, functionally distinct regulatory elements interact to generate complex expression patterns in mammals