(A) Sequence logo displaying nucleotide position biases of seed sequences (guide positions 2–8) with the lowest off-target score in siSPOTR analysis (top 1%). (B) Sequence logo displaying position biases in sequences from a that harbor a T in guide position 2. (C) Knockdown efficiency of ARTi-shRNAmirs. Flow cytometric quantification of GFP knockdown efficiency in immortalized mouse embryonic fibroblastss (MEFs) 2 d after transduction with indicated ARTi-shRNAmir or control short-hairpin RNAs (shRNAs). Percent knockdown is normalized to shRen.713 control. shRen.660 served as a neutral control whose target site was not included in the reporter. Red asterisks indicate ARTi-shRNAmirs that were selected for follow-up studies. (D) Toxicity of ARTi-shRNAmirs. Competitive proliferation assays of three murine cell lines after transduction with ARTi-shRNAmirs, shRen.713, or shMyc.1834 control, showing the relative fraction of shRNA-expressing cells at indicated time points following the initial measurement (day 4 after shRNA transduction). (E) Principal component analysis of gene expression profiling in HT-1080 cells. Respective shRNAmirs and treatments are indicated in the respective colors. X-axis: principal component 1; Y-axis: principal component 2. (F) Volcano plots visualizing de-regulated genes in HT 1080 cells. All shRNAmirs and treatments were tested against the empty vector control. X-axis: -log10(p-value); Y-axis: log2 fold change. (G) Cell growth assay for ARTi-shRNAmir transduced cells and their parental controls in the presence and absence of dox. ARTi, artificial RNA interference; shRNAmirs, micro-RNA embedded shRNAs.