RNA interference (RNAi) provides a powerful tool to elucidate gene function by silencing the expression of a specific gene at the RNA level. The recently discovered CRISPR-Cas9 bacterial immune system can be repurposed to easily create gene mutations and replacements in the mammalian genome and has revolutionized the field of genome engineering and reinforced the field of gene therapy research. Therefore, the RNAi and CRISPR-Cas9 approaches that enable the selective modification of gene expression make them intriguing therapeutic reagents for combating HIV and other infectious diseases. Delivery of RNAi and CRISPR-Cas9 components to the target cells is commonly achieved by use of a DNA vector encoding the effectors. The accurate expression of designed components is critical for the proper and specific execution of their function. In this thesis we focus on studying transcription as mediated by type 3 Pol III promoters and, based on the acquired Pol III knowledge, we develop modified and novel Pol III transcription systems to express optimized RNAi and CRISRP-Cas reagents
