Development of clinical CRISPR/Cas gene editing therapeutics requires a guide RNA with specificity to a region in the target gene, and an endonuclease termed Cas (CRISPR-associated) that mediates RNA-guided specific DNA cutting. Each CRISPR/Cas system has specific functional requirements that limit their potential clinical utility. The Cas9 endonucleases derived from S. pyogenes or S. aureus have been utilized in pre- clinical studies and are being developed for clinical trials in humans. However, both S. pyogenes and S. aureus are common human pathogens, resulting in potential adaptive immunity to Cas9 (1,2). This pre-existing immunity may limit the clinical efficacy of gene editing therapeutics based on Cas9 in a subset of patients, especially in situations involving viral vectors or repeated infusions of Cas9. These, and other limitations of specific Cas endonucleases, argue for the development of a diverse armamentarium of gene editing enzymes. Excision BioTherapeutics is specialized in developing novel Cas therapeutics for targeting human viral pathogens. Recently, several novel Cas enzymes, termed CasX (Cas12e) and CasY (Cas12d), were identified in metagenomic sequencing data from environmental isolates (3) and are being actively developed by Excision BioTherapeutics for their unique potential for clinical application. These novel Cas? are from non-pathogenic bacterial species, are smaller than spCas9, and have distinct protospacer adjacent motif (PAM) requirements. Therefore, in the current application we will leverage our expertise in CRISPR therapeutics to accelerate the pre-clinical development of these therapeutically favorable, novel enzymes. To do so, we will synthesize engineered genes encoding CasX and CasY homologs that are optimized for both recombinant protein production and use in human cells. Through an established recombinant Cas production pipeline, we will express, purify, and validate the activity of CasX and CasY homologs. In vitro assays will be used to optimize gRNA format and cleavage conditions. Using a combination of in vitro and in vivo assays, we will then perform a direct comparison of CasX1 and CasY1 with Cas9 in cleaving the HIV-1 provirus. Finally, next generation sequencing approaches will be used to assess the relative efficiency and specificity of the enzymes in vitro and in HIV-1 infected human cells. Ultimately, our approaches will lead to the development of a panel of novel, compact CRISPR/Cas therapeutics that can be administered to humans without potential interference from pre-existing immunity.