Designing Hairpin-Based RNAi Shuttles

The intrinsic potency of a hairpin-based silencing trigger is one of the most critical determinants for applications such as the creation of stable knockdown cell lines or transgenic knockdown animals. To facilitate the creation of potent silencing constructs, we systematically investigated various parameters for their impact on the knockdown efficacy of hairpin-based RNAi shuttles. Results from our investigation clarified several misleading claims in the literature and defined a set of optimal parameters for hairpin-based RNAi shuttles. In this chapter, we will discuss various factors, such as scaffold (shRNA or miR30-based scaffold), configuration (length of stem and loop size), and target sequence selection, influencing the potency of silencing constructs and provide practical guidelines for designing effective hairpin-based RNAi shuttles.