Abstract Numerous genetic variants associated with inherited platelet function disorders are classified as variants of unknown significance (VUS) because they lack functional evaluation. The functional validation of the genetic causes of platelet mediated disease has been difficult because of the inability to make precise genetic modifications in anucleate human platelets. As the precursor to platelets, megakaryocytes share many functions with platelets and are the natural choice for platelet function studies, but have been traditionally difficult to genetically modify . In this proposal, we develop a non-viral and selection free CRISPR/CAS9 approach to make precise gene edits in human cord blood and adult CD34+ cell derived megakaryocytes using homology directed repair. We apply the approach to functionally and mechanistically define VUS in ITGA2B, mutations in which can cause the bleeding disorder Glanzmann’s Thrombasthenia. Our preliminary data demonstrate the precise generation of insertions and point mutations in the gene ITGA2B in >95% of megakaryocytes. We show that megakaryocytes harboring point mutations or insertions in ITGA2B that are known to cause Glanzmann’s mimic functional responses observed for platelets from patients. In Aim 1 we generate 57 VUS or likely pathogenic variants in ITGA2B and test their effect on platelet-like functional responses in megakaryocytes towards their clinical reclassification, while at the same time defining rules for efficient homology directed repair in megakaryocytes. We further deep phenotype and mechanistically dissect select ITGA2B variants, including in human platelets generated in mice. In Aim 2, we use homology directed saturating mutagenesis in CD34+ derived megakaryocytes, followed by high throughput sequencing, to identify all functional amino acid changes across mutation hot-spots of ITGA2B. This work is innovative: we use a novel and straightforward approach to generate precise point mutations and insertions at unprecedented levels in human CD34+ cells differentiated into megakaryocytes. We use innovative methods, including the generation of gene edited human platelets in mice, and saturating mutagenesis functional screens in primary cells, to examine the effect of genetic variants on megakaryocyte/platelet functions. This work is significant because it will result in the clinical classification of genetic variants that can be directly applied for the genetic diagnosis of patients, and provide hope for future treatment options. Our studies will also provide new mechanistic insights into how variants affect ITGA2B production and function in a physiologically relevant human primary cell.