Evaluation of RNAi and CRISPR technologies by large-scale gene expression profiling in the Connectivity Map
Fig 1
RNAi reagents have widespread off-target effects.
(A) Heat map of Spearman correlations among pairs of shRNAs targeting control genes. Correlation on the diagonal reveals a gene expression signal that is reproducible and specific to each shRNA, despite the absence of a target. Control genes are labeled as follows: GFP, LUC, RFP, and LAC. Additional control treatments are grouped under Ctrl; 1: pgw, a lentivirus with no U6 promoter and no shRNA; 2: empty_vector, a lentivirus with a run of 5 thymidines immediately after the U6 promoter, to terminate transcription; 3: UnTrt, wells that did not receive any lentivirus. (B) Distribution of pairwise correlations of shRNA signatures with the same gene target, the same 6- and 7-mer seed sequence, and all pairs of shRNAs. Data shown are from HT29 cells. Pairs of shRNAs with the same seed correlate much better than those with the same gene, which correlate only marginally better than random pairs. (C) The fraction of pairs of shRNA signatures with the same target gene (red) or the same 6-mer seed (blue) that are statistically significant (q < 0.25) in each cell line. In all cell lines, correlation due to seed is more often significant than correlation due to gene. See S2 Data. Ctrl, control; GFP, green fluorescent protein; LAC, beta-galactosidase; LUC, firefly luciferase; pgw, puromycin-GFP-WPRE; RFP, red fluorescence protein; RNAi, RNA interference; shRNA, short hairpin RNA; U6, human U6 polymerase III promoter; UnTrt, untreated.