Mutational analysis has long been a valuable tool for deciphering gene function. However, systematic repeated targeting of a single locus is difficult and is not a routine approach in multicellular organisms. Yikang Rong and colleagues at the National Cancer Institute have developed the Site-specific Integrase mediated Repeated Targeting (SIRT) method to facilitate targeted mutagenesis in Drosophila melanogaster. SIRT targets a landing site for the phage phiC31 integrase and allows the generation of several genetic variants at a locus of interest without having to perform multiple experiments. SIRT requires the construction of a series of plasmid vectors with varying arrangements of DNA elements. By taking advantage of bacterial recombineering approaches, SIRT bypasses the shortcomings of traditional cloning techniques that rely on the availability of convenient restriction enzyme cut sites. SIRT Combines Homologous Recombination, Site-Specific Integration, and Bacterial Recombineering for Targeted Mutagenesis in Drosophila, is one of June’s featured articles in Cold Spring Harbor Protocols. Like all of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.