(i) Wild-type AAV genome. The 2 AAV genes (rep and cap, encoding replication and capsid proteins) are flanked by inverted terminal repeats (ITRs) that serve as replication and packaging signals. (ii) In a conventional single-stranded AAV vector genome, rep and cap are replaced by an shRNA expression cassette. A stuffer sequence is needed to increase the genome size to the packaging optimum. (iii) In an advanced double-stranded AAV vector, the total size of shRNA and stuffer is reduced to less than half the size of wild-type AAV, and 1 ITR is partially deleted (indicated by asterisk). As a result, the vector undergoes a single replication cycle in cells during virus production, leading to duplication of the shRNA/stuffer cassette. In infected cells, the 2 shRNA copies rapidly anneal with each other, which results in instant and robust RNAi expression. (iv) Alternatively, the stuffer DNA can be replaced with further copies of the same (or other) shRNA cassettes to potentiate the RNAi effect or to create a coRNAi vector directed against multiple targets.