Journal: Genome Biology
Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries
Figure Lengend Snippet: A pooled screen for functional validation of CLD. a The screening strategy in SW480 cells. In brief, a pool of mutant SW480 cells harboring 12,471 sgRNA designs against 408 genes was generated by lentiviral infection and antibiotic selection. Fourteen million cells per condition were then treated with PBS (control) or recombinant TRAIL (treatment) for a total of 12 days. Subsequently, the genomic DNA of the samples was extracted and sgRNA composition analyzed by next-generation sequencing ( NGS ). b Comparison of sgRNA sequence counts between two biological replicates demonstrates high reproducibility (Pearson correlation coefficient ~0.79). c Distribution of sgRNAs targeting positive pathway regulators ( CASP8 , CASP3 , FADD , BAX , BID , TNFRSF10A , TNFRSF10B ) in red , negative regulators ( XIAP , BCL2L1 ) in blue , and random, non-targeting sgRNAs in orange between TRAIL ( y-axis ) and PBS ( x-axis ) treated cell populations. d Scatter plot showing relative enrichment of genes ( y-axis ) with their corresponding p value ( x-axis ). Positive regulators are plotted in red , negative regulators in blue , and random, non-targeting sgRNAs in orange. P values were calculated by Wilcoxon rank sum test between 30 sgRNAs of one gene and 200 random, non-targeting sgRNAs. Log 2 fold change is calculated as median log 2 ratio between normalized sgRNA count of TRAIL- over PBS-treated populations. The vertical line marks a p value of 0.05. e – g Median normalized fold change of all sgRNAs targeting three essential TRAIL pathway components. A total of 100 sgRNAs are depicted for each gene. Enriched sgRNAs are colored in red , depleted sgRNAs in grey . The dashed line represents the median fold change of all sgRNAs of the corresponding gene
Article Snippet: The purified libraries were controlled for correct size using DNA High Sensitivity Assay on a BioAnalyzer 2100 (Agilent) and then sequenced on a MiSeq (Illumina) by 100-bp single-end sequencing and addition of 20 % PhiX Control v3 (Illumina) at a concentration of 8 pM.
Techniques: Functional Assay, Mutagenesis, Generated, Infection, Selection, Recombinant, Next-Generation Sequencing, Sequencing