ABSTRACT The pathogenic ameba Entamoeba histolytica is a deadly human pathogen with limited therapeutic options. Entamoeba causes dysentery and liver abscesses in 50 million people annually but despite being an important human pathogen, the mechanisms of pathogenesis are poorly understood. Over the last several years a number of genetic advances in Entamoeba have improved the study of virulence factors and increased our understanding of amebic biology. However, the full adaptation of the state of the art genetic approaches are still lacking in Entamoeba and all efforts to genetically manipulate the genome have been unsuccessful to date (i.e. homologous recombination or restriction enzyme mediated recombination). We propose to adapt the highly utilized CRISPR technology to Entamoeba. Application of a CRISPR approach will be a significant advance in the field and be an important contribution to the scientific community working on this and related parasite systems. We have strong data that CRISPR technology can be functional in ameba. We demonstrate that (i) Cas9 regulated expression is important to avoid cellular toxicities, (ii) we can achieve good expression levels of guide RNA, and (iii) an episomal plasmid consisting of a mutated luciferase reporter gene can be corrected by Cas9-mediated genome editing to restore luciferase signal. With these data in hand, our goal is now to advance the methodology by increasing its efficiency and adapting it for use in genome editing with the goal of having a robust technology that will be able to identify important biological insights using genetic approaches. Our workflow is: Aim 1. Prove episomal recombination in our current system by sequence analysis. Additionally optimize efficiency of editing by a variety of approaches (increase guide RNA expression, use preassembled Cas9/guideRNA ribonucleoprotein complex, use CRISPR/Cpf2 to target AT-rich PAM sequences, and increase functional levels of Cas9). Once episomal genetic targeting has been optimized, we will proceed with Aim 2: Use highest efficiency approach identified in Aim 1 to mediate genome editing by (i) disrupting a gene previously shown to be amenable to genetic downregulation, (ii) Myc or GFP-tagging a gene, (iii) target luciferase to random genomic regions, and (iv) demonstrating drug susceptibility of parasites to Fumagillin (MetAP2 pathway). Development of CRISPR genome editing in Entamoeba will be an important genetic breakthrough in the system and will expand studies of biological pathways relevant to virulence and drug susceptibilities. This work is suitable for an R21 mechanism as success with this important tool development technology will provide an important resource for the scientific community.