Designer nucleases allow for the precise modification of a given DNA sequence by the introduction of a sequence-specific double strand break. This targeted genetic engineering confers the ability to modify genomes of complex organisms, and has far-reaching applications in human medicine, agriculture, and biotechnology. As these nucleases act in a sequence specific manner, understanding their specificity is of paramount importance to prevent potentially genotoxic side effects. In this thesis, I assessed the ability of a class of designer nucleases (ZFNs)--zinc finger nucleases--to simultaneously inactivate two genes encoding entry factors required for HIV infection in human CD4 T cells. Additionally, I sought to develop a high-throughput means of identifying sites of designer nuclease off-target activity across the genome, in an effort to better understand the factors governing designer nuclease specificity. This work demonstrates the ability of ZFNs to simultaneously modify two distinct genetic loci in primary human CD4 T cells--the main target of HIV infection. These gene-modified cells are protected from HIV infection and represent a novel means of treating--and potentially curing HIV infection. This work also demonstrates that DNA double-strand breaks introduced by a single designer nuclease at on- and off-target loci can result in the formation of genomic rearrangements. Taken together, this work advances in the field of genome engineering on two fronts--a novel therapeutic application of designer nucleases and a novel means of detecting off-target genomic modification.