Journal: bioRxiv

Article Title: Scalable pooled CRISPR screens with single-cell chromatin accessibility profiling

doi: 10.1101/2020.11.20.390971

Figure Lengend Snippet: ( a ) Chromatin remodeling complex subunits and cofactors targeted in the CRISPR library. ( b ) Heatmap of chromatin accessibility Z -scores at transcription factor binding sites (TFBSs) for the different chromatin remodeling complexes targeted in the screen. We converted the fraction of accessible regions for each TFBS into Z -scores (using all cells in the screen). For visualization, we first average over all cells for a particular target gene and then average over all genes in the complex. The histograms ( left ) show the distribution of Z -scores for each complex. The FACT complex is not shown due to a low number of single cells ( n = 75 cells). ( c ) UMAP representation of the genes perturbed in the screen based on the TFBS differential accessibility Z -score profiles. Subunits of the SWI-SNF pBAF complex are labeled with filled circles and gene names. ( d ) The number of transcription factor binding sites with significant differential accessibility for cells that receive a specific genetargeting CRISPR perturbation, as compared to cells that receive a non-targeting (NT) control sgRNA (FDR q ≤ 0.1). SWI/SNF components and co-factors are highlighted in red. ( e ) The percent of ATAC fragments in enhancers and promoters in cells transduced with ARID1A -targeting and NT sgRNAs. Each point is a single cell. K562 enhancer and promoter genome segmentation is from ENCODE (see Methods ). ( f ) CRISPR-targeted chromatin complex genes with significant differential accessibility at enhancers and/or promoters. ( g ) Volcano plots showing significant changes in accessibility at TFBSs in cells transduced with ARID1A (left), SMARCA5 ( middle ) and RCOR1 ( right ) -targeting sgRNAs. Standardized Z -scores are averaged over single cells. Points in red represent TFBSs with a significant change in accessibility (FDR q ≤ 0.1 and | Z -score| > 0.25).

Article Snippet: To generate NIH-3T3 and HEK293FT cells expressing single-guide RNAs (sgRNAs) for the human/mouse experiment, 10 human non-targeting sgRNAs and 10 mouse non-targeting sgRNAs ( Supplementary Table 2 ) were individually synthesized and cloned into the lentiviral transfer vector CROPseq-Guide-Puro (Addgene 86708), which leads to the synthesis of an RNA Pol3 transcript of the Cas9 sgRNA and an RNA Pol2 polyadenylated transcript containing the puromycin resistance gene, a U6 promoter, and the sgRNA.

Techniques: CRISPR, Binding Assay, Labeling, Control, Transduction

Journal: bioRxiv

Article Title: Scalable pooled CRISPR screens with single-cell chromatin accessibility profiling

doi: 10.1101/2020.11.20.390971

Figure Lengend Snippet: ( a ) Heatmap of chromatin accessibility Z -scores at histone and DNA modifications for different CRISPR perturbations ( n = 3 sgRNA per gene). We converted the fraction of accessible regions for each modification into Z -scores (using all cells in the screen). For visualization, we show the average Z -score for all cells receiving a particular sgRNA. ( b ) Distances in the histone and DNA modifications accessibility profiles shown in panel a between sgRNAs targeting different genes and sgRNAs targeting the same gene. The distance metric is 1-(Pearson correlation of the Z -scores). ( c ) Pearson correlation between averaged accessibility Z -scores at histone and DNA modifications of the indicated number of single cells and the average profile of 400 single cells, for cells with either EZH2 -targeting or non-targeting (NT) sgRNAs. ( d ) UMAP representation of chromatin accessibility Z -scores at histone and DNA modifications from single cells receiving either EZH2 or NT sgRNAs. Also shown is the same UMAP representation with single cells colored by TFBS accessibility enrichment scores for CBX2, CBX8, EZH2, POLR2B , and SIRT6 . ( e ) H3K27me3 ChIP-seq coverage at the HOXA-D loci ( top ). Changes in accessibility at the HOXA-D loci in cells transduced with EZH2 -targeting or NT sgRNAs ( bottom ). *** denotes p < 0.001. ( f ) CRISPR-sciATAC fragments mapping to the HOXA locus in cells transduced with EZH2 -targeting or NT sgRNAs ( n = 510 cells per condition). The sum of all ATAC fragments over the entire HOXA locus in cells transduced with EZH2 -targeting and NT sgRNAs is shown on the right. K562 H3K27me3 ChIP-seq coverage is shown at the bottom. ( g ) Gene expression (qPCR) of EZH2, HOXA3, HOXA5, HOXA11A, HOXA13 and HOXD9 for cells transduced with either EZH2 -targeting or NT sgRNAs

Article Snippet: To generate NIH-3T3 and HEK293FT cells expressing single-guide RNAs (sgRNAs) for the human/mouse experiment, 10 human non-targeting sgRNAs and 10 mouse non-targeting sgRNAs ( Supplementary Table 2 ) were individually synthesized and cloned into the lentiviral transfer vector CROPseq-Guide-Puro (Addgene 86708), which leads to the synthesis of an RNA Pol3 transcript of the Cas9 sgRNA and an RNA Pol2 polyadenylated transcript containing the puromycin resistance gene, a U6 promoter, and the sgRNA.

Techniques: CRISPR, Modification, ChIP-sequencing, Transduction, Gene Expression