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. Overall design: EGFRdel19::dsRed::ARTi engineered PC-9 cells harboring the Dox-inducible ARTi-shRNAmir were treated with Dox to elicit the knock-down of the EGFR transgene. KRASG12R-ARTi engineered MIA PaCa2 cells harboring the Dox-inducible ARTi-shRNAmir were treated with Dox to elicit the knock-down of the KRAS transgene. Differences in gene expression were monitored.