RNA-interference (RNAi) is ubiquitously used as a research tool, with dozens of candidates using this technology currently in FDA clinical trials. A particular segment of the RNAi world, short hairpin RNAs (shRNAs) seeks to enable stable, long-term reduction of a target protein. Here, we present an optimization of shRNA structure that allows increased reduction of targeted proteins as unwanted processing events are bypassed, increasing the amount of mature RNAi species created. In Chapter 1, we present current literature as it pertains to RNAi biogenesis, regulation, and optimization of shRNA effectiveness. In Chapter 2, we present our findings of extensive shRNA precursor degradation, and complete an unbiased screen for structures that bypass this degradation and optimize RNAi activity. In Chapter 3, we offer our observations of negative processing events associated with RNA Polymerase III (RNAPIII)-driven shRNAs, including but not limited to the same extensive precursor degradation. Together, these data present new details in the dysregulation of endogenous miRNA biogenesis by shRNAs, and offer guidance towards more accurate and potent engineered shRNA regulators less likely to induce off-target effects.