Sequence-specificity is the key to effective genetic targeting. With specificity, targeted genes can be manipulated in multiple ways; without it, gene therapy agents become loose canons within cells. Triplex forming oligonucleotides (TFOs) bind in the major groove of duplex DNA with high specificity and affinity. Because of these characteristics, TFOs have been proposed as homing devices for genetic manipulation in vivo. Here we review work demonstrating the ability of TFOs and related molecules to alter gene expression and mediate genome modification in mammalian cells. Recent studies have established that TFOs can mediate targeted gene knock out in mice, laying the foundation for the potential application of these molecules in human gene therapy.