Precision RNAi using synthetic shRNAmir target sites

Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient, inducible, and reversible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects. Quant-seq of RKO and HT-1080 human cell lines transduced with ARTi.6570, ARTi.6634, ARTi.6786 artificial short-hairpin RNA constructs or with empty vector control (+/- trametinib treatment) in biological triplicates.