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Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo., The molecular dynamics associated with neuronal activation patterns in vivo are unclear. Lacar et al. perform single-nuclei RNA-sequencing of hippocampal neurons from mice exposed to a novel environment, and identify large-scale transcriptome changes in individual neurons associated with the experience.

Single-cell transcriptomic assays have enabled the de novo reconstruction of lineage differentiation trajectories, along with the characterization of cellular heterogeneity and state transitions. Several methods have been developed for reconstructing developmental trajectories from single-cell transcriptomic data, but efforts on analyzing single-cell epigenomic data and on trajectory visualization remain limited. Here we present STREAM, an interactive pipeline capable of disentangling and visualizing complex branching trajectories from both single-cell transcriptomic and epigenomic data. We have tested STREAM on several synthetic and real datasets generated with different single-cell technologies. We further demonstrate its utility for understanding myoblast differentiation and disentangling known heterogeneity in hematopoiesis for different organisms. STREAM is an open-source software package., The increasing accessibility of single cell omics technologies beyond transcriptomics demands parallel advances in analysis. Here, the authors introduce STREAM, a pipeline for reconstruction and visualization of differentiation trajectories from both single-cell RNA-seq and ATAC-seq data.

Current technologies for upregulation of endogenous genes use targeted artificial transcriptional activators but stable gene activation requires persistent expression of these synthetic factors. Although general "hit-and-run" strategies exist for inducing long-term silencing of endogenous genes using targeted artificial transcriptional repressors, to our knowledge no equivalent approach for gene activation has been described to date. Here we show stable gene activation can be achieved by harnessing endogenous transcription factors ( EndoTF s) that are normally expressed in human cells. Specifically, EndoTFs can be recruited to activate endogenous human genes of interest by using CRISPR-based gene editing to introduce EndoTF DNA binding motifs into a target gene promoter. This Precision Editing of Regulatory Sequences to Induce Stable Transcription-On ( PERSIST-On ) approach results in stable long-term gene activation, which we show is durable for at least five months. Using a high-throughput CRISPR prime editing pooled screening method, we also show that the magnitude of gene activation can be finely tuned either by using binding sites for different EndoTF or by introducing specific mutations within such sites. Our results delineate a generalizable framework for using PERSIST-On to induce heritable and fine-tunable gene activation in a hit-and-run fashion, thereby enabling a wide range of research and therapeutic applications that require long-term upregulation of a target gene.

CRISPR prime editing offers unprecedented versatility and precision for the installation of genetic edits in situ . Here we describe the development and characterization of the Multiplexing Of Site-specific Alterations for In situ Characterization ( MOSAIC ) method, which leverages a non-viral PCR-based prime editing method to enable rapid installation of thousands of defined edits in pooled fashion. We show that MOSAIC can be applied to perform in situ saturation mutagenesis screens of: (1) the BCR-ABL1 fusion gene, successfully identifying known and potentially new imatinib drug-resistance variants; and (2) the IRF1 untranslated region (UTR), re-confirming non-coding regulatory elements involved in transcriptional initiation. Furthermore, we deployed MOSAIC to enable high-throughput, pooled screening of hundreds of systematically designed prime editing guide RNA ( pegRNA ) constructs for a large series of different genomic loci. This rapid screening of >18,000 pegRNA designs identified optimized pegRNAs for 89 different genomic target modifications and revealed the lack of simple predictive rules for pegRNA design, reinforcing the need for experimental optimization now greatly simplified and enabled by MOSAIC. We envision that MOSAIC will accelerate and facilitate the application of CRISPR prime editing for a wide range of high-throughput screens in human and other cell systems.