20 nt target specific sgrna sequences  (CustomArray Inc)

Journal: Nature biotechnology

Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks

doi: 10.1038/nbt.4062

Figure Lengend Snippet: a , Schematic of genome-scale CRISPRa screening approach (see text for details). b , Overview of CRISPRa screen results. Negative τ values indicate depletion and positive values enrichment of cells following imatinib selection. Significant candidate genes (FDR<0.05, p<0.001) are in colour (blue = depleted, red = enriched). Validated candidate genes are labelled in black. Mann-Whitney U test was used to calculate p-values as described previously . To correct for multiple hypothesis testing, we first performed random sampling with replacement among the set of τ values for non-targeting control sgRNAs and calculated p-values for each sampling. Then, we calculated the false discover rate (FDR) based on the distribution of p-values for all genes in the library and for non-targeting controls generated above. c, Candidate gene validation. Enrichment of candidate sgRNA expressing cells was measured over time. Values represent the mean of three different sgRNAs targeting each gene with s.e.m. Grey shading = two standard deviations of sgNTCs at day 15. All values from separate sgRNAs on days 7, 11 and 15 normalised to baseline or untreated cells are shown in .

Article Snippet: For the CRISPRa library, the designed 20 nt target specific sgRNA sequences were synthesised as a pool, on microarray surfaces (CustomArray, Inc.), flanked by overhangs compatible with Gibson Assembly into the pSico based sgLenti sgRNA library vector (see for vector map).

Techniques: Selection, MANN-WHITNEY, Sampling, Generated, Expressing

Journal: Nature biotechnology

Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks

doi: 10.1038/nbt.4062

Figure Lengend Snippet: a, Concept of the application of the orthogonal system for directional gene interaction studies. In the same cell, one gene is activated (CRISPRa) while another gene in knocked out (SaCas9 nuclease). b–d, Correlation of τ values from two clonal cell line replicates is shown for b, gene activation, c, gene knockout and d, all possible combinations thereof. Correlation values (r) are Pearson product-moment correlation coefficients. e , Schematic of perturbation data set from each gene pair (blue = depleted, red = enriched, NTC = non-target control sgRNA) f, Formula for calculating Ψ scores. Negative Ψ scores define interactions in which directionality could be inferred. g , To determine which of both interaction partners acts up- or downstream, τ activation values were multiplied with genetic interaction scores. Positive values indicate a downstream function, negative values an upstream function of the activated gene. h , Based on GI and Ψ scores determined by the full orthogonal interaction screen, a genetic interaction model was constructed. For positive regulators of cell fitness, nodes are shown in red and negative regulators in blue. Arrow-shaped edges indicate inferred directional interactions between nodes. Line-shaped edges symbolise genetic interactions where directionality could not be inferred. Node sizes are proportional to the degree of connectivity. In total, 2258 gene:gene combinations that passed the filter criteria were considered for the construction of the directional genetic interaction network.

Article Snippet: For the CRISPRa library, the designed 20 nt target specific sgRNA sequences were synthesised as a pool, on microarray surfaces (CustomArray, Inc.), flanked by overhangs compatible with Gibson Assembly into the pSico based sgLenti sgRNA library vector (see for vector map).

Techniques: Activation Assay, Gene Knockout, Construct