Journal: Genome Biology

Article Title: Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens

doi: 10.1186/s13059-024-03176-z

Figure Lengend Snippet: A Schematic of the CRISPRi protocol in primary CD4 + T cells. B Histograms showing expression of the target gene ( CD4 , CD81 , BST2 ) 10 days after gRNA transduction into primary CD4 + T cells expressing a CBh-ZIM3-dCas9 repressor construct, analyzed by flow cytometry. gRNA #1 and gRNA #2 refer to two different gRNA designs for a given TSS. The wild-type (WT) control are non-transduced cells stained with the same antibody for the corresponding target gene. C Quantification of the percentage of cells retaining cell surface expression of CD4 , CD81 , and BST2 at days 4, 6, 8, or 11 after transduction of a TSS-targeting gRNA (red) or NT control gRNA (gray) into primary CD4 + T cells expressing CBh-ZIM3-dCas9, analyzed by flow cytometry. Replicates are cells derived from four donors. Differences between non-targeting and targeting gRNAs are significant for all genes and timepoints ( p -value < 0.00005, Bonferroni-Dunn test). D Normalized expression levels of the same target genes, measured by 10X Genomics 3′ scRNA-seq, 11 days after the corresponding targeting (red) or non-targeting (gray) gRNAs were transduced into primary CD4 + T cells expressing a CBh-ZIM3-dCas9 repressor construct. The dashed line indicates the median expression level in cells with non-targeting controls. The number of cells in each group is indicated at the top. Note gRNA #1 and #2 for CD81 TSS were analyzed together due to sequence similarity

Article Snippet: gRNA oligos were synthesized, PCR amplified, and cloned into the CRISPRi CROPseq backbone using Gibson assembly, as previously described [ ], by VectorBuilder.

Techniques: Expressing, Transduction, Construct, Flow Cytometry, Control, Staining, Derivative Assay, Sequencing