Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of "chromatin hubs"-they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.

In Brief A highly multiplexed CRISPRi screen uncovers gene-enhancer relationships at scale.

At a dose as low as 1 microgram per kilogram of body weight, lysergic acid diethylamide (LSD) significantly decreased the suppressive effect of electric shock on licking behavior of the rat. Attenuation of punishment was also obtained with mescaline, but neither dimethyltryptamine nor delta9-tetrahydrocannabinol was active in this test. Cyproheptadine and alpha-propyldopacetamide, drugs that interfere with the function of neurons that contain serotonin, have a behavioral effect similar to that of LSD and mescaline, which suggests that the attenuation of punishment produced by these hallucinogens may result from decreased activity of such neurons.

High-throughput chemical screens typically use coarse assays such as cell survival, limiting what can be learned about mechanisms of action, off-target effects, and heterogeneous responses. Here, we introduce “sci-Plex,” which uses “nuclear hashing” to quantify global transcriptional responses to thousands of independent perturbations at single-cell resolution. As a proof of concept, we applied sci-Plex to screen three cancer cell lines exposed to 188 compounds. In total, we profiled ~650,000 single-cell transcriptomes across ~5000 independent samples in one experiment. Our results reveal substantial intercellular heterogeneity in response to specific compounds, commonalities in response to families of compounds, and insight into differential properties within families. In particular, our results with histone deacetylase inhibitors support the view that chromatin acts as an important reservoir of acetate in cancer cells.

SUMMARY To study the development of the human retina, we use single-cell RNA sequencing (RNA-seq) at key fetal stages and follow the development of the major cell types as well as populations of transitional cells. We also analyze stem cell (hPSC)-derived retinal organoids; although organoids have a very similar cellular composition at equivalent ages as the fetal retina, there are some differences in gene expression of particular cell types. Moreover, the inner retinal lamination is disrupted at more advanced stages of organoids compared with fetal retina. To determine whether the disorganization in the inner retina is due to the culture conditions, we analyze retinal development in fetal retina maintained under similar conditions. These retinospheres develop for at least 6 months, displaying better inner retinal lamination than retinal organoids. Our single-cell RNA sequencing (scRNA-seq) comparisons of fetal retina, retinal organoids, and retinospheres provide a resource for developing better in vitro models for retinal disease.

Integrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We profiled epithelial cells undergoing a spontaneous, spatially determined EMT in the presence or absence of TGF-β via single-cell RNA-seq. Pseudospatial trajectory analysis identified continuous waves of gene regulation, as opposed to discrete "partial" stages of EMT. KRAS was connected to exit from the epithelial state and acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along EMT. Inhibiting the KRAS effector MEK, and its upstream activators EGFR and MET, demonstrates that interruption of key signaling events reveals regulatory "checkpoints" in the EMT continuum that mimic discrete stages and reconciles opposing views of the program that controls EMT. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Several groups recently coupled CRISPR perturbations and single-cell RNA-seq (scRNA-seq) for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to ~50% swapping of guide RNA-barcode associations due to lentiviral template switching. We optimize a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, confirming this strategy performs robustly and doubling the rate at which guides are assigned to cells to 94%.

Several groups recently reported coupling CRISPR/Cas9 perturbations and single cell RNAseq as a potentially powerful approach for forward genetics. Here we demonstrate that vector designs for such screens that rely on cis linkage of guides and distally located barcodes suffer from swapping of intended guidebarcode associations at rates approaching 50% due to template switching during lentivirus production, greatly reducing sensitivity. We optimize a published strategy, CROPseq, that instead uses a Pol II transcribed copy of the sgRNA sequence itself, doubling the rate at which guides are assigned to cells to 94%. We confirm this strategy performs robustly and further explore experimental best practices for CRISPR/Cas9based single cell molecular screens.