Red box: Preferred setting (due to highest specificity and potency) for therapeutic RNAi, i.e., perfect RNAi duplexes against perfect targets. As shown, this is also the trigger-target combination that is most prone to saturating Ago-2 and thus causing cytotoxicity. As further implied by our data, this risk may be lowered by reducing the homology either between the two dsRNA strands or to the target. Hence, imperfect triggers against imperfect targets were least dependent on Ago-2. Yet they may also be least useful for human RNAi therapies. We thus favor two alternative strategies that both aim at avoiding saturation and thus alleviating shRNA toxicity and for which we have provided proof-of-concept in this article. One (a) is to transiently overexpress (e.g., using AAV vectors) limiting cellular key RNAi components, especially Xpo-5 and Ago-2. This should for instance benefit cancer-directed RNAi gene therapies, where instant and maximally potent target knockdown might be critical. It should likewise improve functional in vitro RNAi screens, where short-term and robust siRNA or shRNA activity is typically desired. An alternative (but not exclusive) second strategy (b) is to utilize minimal yet effective trigger doses in humans, by employing weak and/or tissue-specific promoters for shRNA expression. Based on our mouse data, we predict that this can also mitigate saturation-induced toxicity and prevent vector loss and thus enhance both efficacy and persistence of RNAi. Accordingly, this specific strategy should be particularly useful for long-term RNAi gene therapies against persisting exogenous targets, such as HIV or hepatitis viruses.