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Coupled single-cell epigenome editing and profiling reveals causal gene regulatory networks [Perturb-ATAC (GBC/sgRNA)]

ABSTRACT: Here we present Perturb-ATAC, a method which combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells, based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by Assay of Transposase-accessible Chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ~3,700 single cells, encompassing more than 75 unique genotype-phenotype relationships.

ORGANISM(S): Homo sapiens

PROVIDER: GSE116285 | GEO | 2018/12/20

REPOSITORIES: GEO

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Coupled single-cell epigenome editing and profiling reveals causal gene regulatory networks [Perturb-ATAC (ATAC)]

Project description:Here we present Perturb-ATAC, a method which combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells, based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by Assay of Transposase-accessible Chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ~3,700 single cells, encompassing more than 75 unique genotype-phenotype relationships.

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Coupled single-cell epigenome editing and profiling reveals causal gene regulatory networks [scATAC-seq]

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Coupled single-cell epigenome editing and profiling reveals causal gene regulatory networks [Bulk ATAC-seq]

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Coupled single-cell epigenome editing and profiling reveals causal gene regulatory networks [Perturb-ATAC (GBC/sgRNA)]

Project description:Coupled single-cell epigenome editing and profiling reveals causal gene regulatory networks [Perturb-ATAC (GBC/sgRNA)]

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Coupled single-cell epigenome editing and profiling reveals causal gene regulatory networks [Perturb-ATAC (ATAC)]

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Perturb-map coupled with spatial transcriptomics identifies mutation associated gene signatures in a mouse model of lung adenocarcinoma

Project description:The cellular architecture of a tumor has a major impact on cancer outcome, and thus there is interest in identifying genes controlling the tumor microenvironment (TME). While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying gene functions operating extracellularly or within a tissue context. To address this, we developed an approach for spatial functional genomics called Perturb-map, which utilizes protein barcodes (Pro-Code) to enable spatial detection of barcoded cells within tissue. We applied Perturb-map to knockout dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Additionally, we paired Perturb-map and spatial transcriptomics for unbiased molecular analysis of Pro-Code/CRISPR lesions. Our studies found in Tgfbr2 knockout lesions, the TME was converted to a fibro-mucinous state and T-cells excluded, concomitant with upregulated TGFb and TGFb-mediated stroma activation, suggesting Tgfbr2 loss on lung cancer cells increased TGFb bioavailability and enhanced its suppressive effects on the TME. These studies establish Perturb-map for functional genomics within a tissue at single cell-resolution with spatial architecture preserved.

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Lineage recording in human cerebral organoids (iTracer-perturb)

Project description:Induced pluripotent stem cell (iPSC) derived organoid systems provide models to study human organ development. Single-cell transcriptome sequencing enables highly-resolved descriptions of cell state heterogeneity within these systems and computational methods can reconstruct developmental trajectories. However, new approaches are needed to directly measure lineage relationships in these systems. Here we establish an inducible dual channel lineage recorder, iTracer, that couples reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze state and lineage relationships in iPSC-derived systems. This data set include the iTracer-perturb data of one cerebral organoid with simultaneous TSC2 perturbation and lineage recording.

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CRISPRai Perturb-seq [Series 1]

Project description:We develop a system for bidirectional epigenetic editing (CRISPRai), in which orthogonal activating (CRISPRa) and repressive (CRISPRi) perturbations are applied simultaneously to multiple loci the same cell. We perform Perturb-seq, a method for single-cell RNA-seq paired with CRISPR guide RNA (gRNA) detection, on cells perturbed using CRISPRai for a panel of pairwise combinations of genes in K562 cells.

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Comparison of dual sgRNA library versus Dolcetto by Perturb-seq

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Compressed Perturb-seq: highly efficient screens for regulatory circuits using random composite perturbations

Project description:Pooled CRISPR screens with single-cell RNA-seq readout (Perturb-seq) have emerged as a key technique in functional genomics, but are limited in scale by cost and combinatorial complexity. Here, we reimagine Perturb-seq’s design through the lens of algorithms applied to random, low-dimensional observations. We present compressed Perturb-seq, in which we measure multiple perturbations per cell or multiple cells per droplet, and decompress these measurements by leveraging the sparse structure of regulatory circuits. Applying compressed Perturb-seq to 598 genes in the immune response to bacterial lipopolysaccharide, we achieve the same accuracy as conventional Perturb-seq at 4 to 20-fold reduced cost, with greater power to learn genetic interactions. We identify known and novel regulators of immune responses and uncover evolutionarily constrained genes with downstream targets enriched for immune disease heritability, including many missed by existing GWAS or trans-eQTL studies. Our framework enables new scales of interrogation for a foundational method in functional genomics.

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