The RNAi library expression system. (A) The Sce* strain expresses the tetracycline repressor (TetR) and T7 phage RNA polymerase (RNAP) for the control of dsRNA expression, while inducible homing endonuclease (I-SceI) cleavage facilitates site-specific RNAi library integration at a locus that has been validated for reproducible and robust inducible expression. (B) RNAi plasmid library constructs replace the I-SceI gene and cleavage site. The RNAi vector consists of opposing T7 promoters regulated by Tet-operators. The RNAi target fragments serve as a template for the production of dsRNA and also provide unique sequence identifiers for each clonal population. (C) Tetracycline induction of dsRNA synthesis. These long (av. ∼600 bp) dsRNAs generate a pool of heterogeneous siRNAs that mediate sequence-specific destruction of the cognate mRNA.