RNA interference has proven to be a powerful tool to inhibit viruses. For the prevention of viral escape, multiple short hairpin RNAs (shRNAs) will have to be employed. This article describes a rapid procedure for the generation of shRNA expression cassettes by parallel cloning as well as a simple strategy for the combination of selected units. After delivery of the shRNA expression cassettes with adeno-associated virus vectors, inhibition of echovirus 30 as well as silencing of an important cellular cofactor of virus replication were achieved. The procedure has the potential to be generally applicable for silencing of multiple endogenous targets or viruses.
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