The characterization of CRISPR/Cas as a method to edit genes that contribute to human diseases provides new opportunities for the development of innovative therapeutic approaches. Seven years ago, this bacterial immune system was adapted to cleave specific regions in the human genome. Currently, there are over a dozen clinical trials planned or in process for cancer, genetic disorders and infectious diseases that utilize CRISPR editing. Moreover, gene editing can be used to cleave and potentially excise viral genomes within infected cells, which offers hope for curative strategies for HIV-1 and hepatitis B infections, as well as others. The majority of pre-clinical and clinical studies use the Cas9 enzyme derived from common human pathogens, and evidence for the existence of humoral and cellular immunity to Cas9 could stymie the utility of this editor in a subset of patients, especially if delivered in vivo using viral vectors, or if therapy requires repeated infusions (1, 2). These and other limitations of the family of Cas9 endonucleases argue for the development of a more diverse toolbox of gene editing enzymes. Recently, several novel Cas enzymes termed CasX (Cas12e) and CasY (Cas12d) were identified in metagenomic sequencing data from environmental isolates (3), and we are actively developing these enzymes for gene editing. The advantages of these enzymes include their derivation from non-pathogenic bacterial species, their relatively compact size compared to Cas9, and their distinct protospacer adjacent motif (PAM) requirements. The cut generated by the guide RNA/CasX ribonucleoprotein complex is asymmetrical and generates single stranded DNA overhangs. This unique cleavage cut could then be leveraged to excise a target region by employing two different guide RNAs that flank the area of interest. By careful selection of guide RNAs, we expect that we can generate DNA overhangs on opposite DNA strands that are complementary to each other so they anneal during DNA repair, essentially excising the intervening region. In addition, we expect that these asymmetrical overhangs can also be used to anneal to an exogenously supplied donor DNA fragment, so that the excised region is replaced by new DNA sequence. Our proposed studies will develop CRISPR/CasX to replace the wild-type CCR5 gene with the CCR5- delta 32 allele in hematopoietic stem cells (HSC) as a next generation approach to the development of a therapeutic application for HIV-1. It is well established that cells from individuals who are homozygous for the CCR5-delta 32 allele are resistant to R5-tropic HIV infection, and generating autologous HSCs carrying two copies of this mutation could potentially allow the permanent cessation of anti-retroviral therapy. To accomplish this goal, we will develop combinations of guide RNAs to excise the CCR5 gene, and replace it with donor DNA carrying the mutant allele. Next generation sequencing approaches (CIRCLE-seq, MTA-seq) will be used to asses...