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.

Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach.

BACKGROUND: X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signaling pathway, and delaying differentiation. RESULTS: To identify the genetic basis of these sex differences, we use a two-step CRISPR screening approach to comprehensively identify X-linked genes that cause the female pluripotency phenotype in murine embryonic stem cells. A primary chromosome-wide CRISPR knockout screen and three secondary screens assaying for different aspects of the female pluripotency phenotype allow us to uncover multiple genes that act in concert and to disentangle their relative roles. Among them, we identify Dusp9 and Klhl13 as two central players. While Dusp9 mainly affects MAPK pathway intermediates, Klhl13 promotes pluripotency factor expression and delays differentiation, with both factors jointly repressing MAPK target gene expression. CONCLUSIONS: Here, we elucidate the mechanisms that drive sex-induced differences in pluripotent cells and our approach serves as a blueprint to discover the genetic basis of the phenotypic consequences of other chromosomal effects.

Leucine-rich repeat-containing G-protein-coupled receptor 5-positive cells in the endometrial stem cell niche.

OBJECTIVE: To study, isolate and characterize leucine-rich repeat-containing heterotrimeric guanine nucleotide-binding protein-coupled receptor 5 (LGR5)-positive cells from human endometrium to determine their functional relevance. DESIGN: Prospective experimental animal study. SETTING: University research laboratories. ANIMAL(S): Nonobese diabetic mice (NOD-SCID) (strain code 394; NOD.CB17-Prkdcscid/NcrCrl). INTERVENTION(S): Human LGR5+ cells were labeled with superparamagnetic iron oxide nanoparticles (SPIOs) and injected under the kidney capsule in immunocompromised mice. MAIN OUTCOME MEASURE(S): Epithelial and stromal LGR5+ cells were isolated from human endometrium by means of fluorescence-activated cell sorting, and phenotypic characterization was performed by means of flow cytometry with the use of hematopoietic and mesenchymal markers. Engrafted SPIO-labeled LGR5+ cells were localized with the use of Prussian blue staining and immunohistochemistry against CD9 and Vimentin. Deep transcriptomic profiling of LGR5+ cells was performed with the use of microarrays and RNA sequencing. RESULT(S): The percentage of LGR5+ cells in human endometrium represented 1.08 ± 0.73% and 0.82 ± 0.76% of total cells in the epithelial and stromal compartments, respectively. LGR5+ cells were phenotypically characterized by abundant expression of CD45 hematopoietic marker and no expression of surface markers CD31, CD34, CD133, CD73, and CD90. Coexpression with the macrophage marker CD163 was detected. Xenotransplantation of labeled LGR5+ cells into the kidney capsules of immunocompromised mice resulted in a weak endometrial reconstitution from this cell of origin. Transcriptomic profiling revealed new attributes for LGR5+ cells related to their putative hematopoietic origin. CONCLUSION(S): These data suggest that endometrial LGR5 is not an endogenous stem cell marker. Instead, LGR5+ cells appear to be recruited from blood to be part of the stem cell niche at the perivascular microenvironment to activate the endogenous niche.

Epigenetic inheritance of cell fates during embryonic development.

During embryonic development a large number of widely differing and specialized cell types with identical genomes are generated from a single totipotent zygote. Tissue specific transcription factors cooperate with epigenetic modifiers to establish cellular identity in differentiated cells and epigenetic regulatory mechanisms contribute to the maintenance of distinct chromatin states and cell-type specific gene expression patterns, a phenomenon referred to as epigenetic memory. This is accomplished via the stable maintenance of various epigenetic marks through successive rounds of cell division. Preservation of DNA methylation patterns is a well-established mechanism of epigenetic memory, but more recently it has become clear that many other epigenetic modifications can also be maintained following DNA replication and cell division. In this review, we present an overview of the current knowledge regarding the role of histone lysine methylation in the establishment and maintenance of stable epigenetic states.