Abstract Number: OC 29.3
Meeting: ISTH 2021 Congress
Background: Human platelets are anucleate and cannot be directly modified using traditional genetic approaches. Instead, human platelet gene function studies depend on mouse models and alternative cell models. As the nucleated precursor to platelets, primary megakaryocytes are the nearest cell to platelets in origin, structure, and function.
Aims: Achieving consistent genetic modifications in primary megakaryocytes has been challenging, and the functional effects on human megakaryoyctes of induced gene deletions for even well studied platelet genes (for example ITGA2B) are unknown. Therefore, our goal was to develop and define the strengths and limitations of a rapid and systematic approach to screen genes for platelet functions in primary megakaryocytes called CRIMSON: CRIspr edited Megakaryocytes for rapid Screening of platelet gene functiON.
Methods: Using CRISPR/CAS9, we achieve efficient non-viral gene editing of a panel of platelet genes in cord blood-derived megakaryocytes without compromising megakaryopoiesis. We use microscopy and flow cytometry analysis to assess platelet-like functional responses in megakaryocytes.
Results: Gene editing induced loss of protein in up to 95% of cells for platelet function genes GP6, RASGRP2, and ITGA2B; for the immune receptor component B2M; and for COMMD7 which was previously associated with cardiovascular disease and platelet function. Gene deletions affected several responses to platelet agonists in megakaryocytes in a manner largely consistent with those expected for platelets. B2M deletion did not significantly affect platelet-like responses, whereas ITGA2B deletion abolished agonist induced integrin activation and spreading on fibrinogen without affecting P-selectin translocation. GP6 deletion abrogated responses to collagen receptor agonists but not thrombin. RASGRP2 deletion impaired functional responses to ADP, thrombin, and collagen receptor agonists. COMMD7 deletion significantly impaired degranulation and integrin activation in response to platelet agonists.
Conclusions: Together, our data recommend CRIMSON for rapid evaluation of platelet gene phenotype associations.
To cite this abstract in AMA style:Montenont E, Bhatlekar S, Jacob S, Kosaka Y, Manne B, Lee O, Parra-Izauierdo I, Tugolukova E, Tolley N, Rondina M, Bray P, Rowley J. CRISPR Edited Megakaryocytes for Rapid Screening of Platelet Gene Functions [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/crispr-edited-megakaryocytes-for-rapid-screening-of-platelet-gene-functions/. Accessed November 29, 2021.
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