Abstract: Site-specific gene modification has the potential to produce permanent changes in genomic DNA to correct genetic defects or to enhance cellular function. But delivery of the molecular tools for site-specific editing in vivo is a major hurdle. In recent years, we have shown that degradable nanoparticles of PLGA can be engineered into surprisingly versatile systems for intracellular delivery of plasmid DNA, siRNA, miRNA, and a variety of synthetic oligonucleotides. Further, we have established several methods for engineering the particle surface to allow for targeting. Now, we have applied this technology to the problem of genome editing, by synthesizing degradable polymer nanoparticles that are loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. These nanoparticles produce site-specific gene editing of human cells in vivo in hematopoietic stem cellengrafted NOD-scid IL2rγnull mice. Intravenous injection of particles containing PNA and DNA produced modification of the human CCR5 gene in hematolymphoid cells throughout the mice, including CD4+ T cells in the spleen, CD34+ hematopoietic stem/progenitor cells (HSPCs) in the bone marrow, colony-forming hematopoietic progenitors, and true hematopoietic stem cells capable of engraftment in a secondary recipient mouse. We also induced specific modification of the human β-globin gene using nanoparticles carrying β-globin-specific targeting molecules and the cystic fibrosis gene using CFTR-specific targeting molecules, demonstrating this method’s versatility. Direct in vivo gene modification, such as we demonstrate here, eliminates the need for cell harvest, providing a mechanism by which to perform gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo.