PB1272 - CRISPR-Based Knockout Red Blood Cells as Tools to Study Red Blood Cell Surface Protein Involvement in Thrombosis

Background: Thrombosis is a vascular disease that involves unwanted clotting of blood within any vessel, resulting in an obstruction. This obstruction is the result of an imbalance in blood coagulation. Venous thrombosis is characterized by a high content of red blood cells (RBCs) in the clot. RBCs contribute largely to the size and stability of a venous clot. However, it remains unclear how RBCs affect clotting. CRISPR/Cas9- mediated genome editing of RBC proteins enables the study of specific proteins and their effect on clot formation in more detail.

Aims: The aim of this study was to develop a new approach to investigate clot formation and fibrinolysis by using CRISPR-based knockout (KO) RBCs.

Methods: Several membrane proteins suspected to play a role in clot formation were targeted and KO cells were generated using CRISPR/Cas9 in hematopoietic stem cells. Growth and differentiation of cultured RBCs was analyzed by flow cytometry. These cultured RBCs were used to form 5 µL clots by incubating the RBCs and human recalcified citrated plasma with tissue factor. Contracting clots were visualized and the fibrin content quantified by confocal microscopy. Moreover, lysis of these clots was measured using the HALO assay.

Results: Our CRISPR approach resulted in KO efficiencies varying from 80-95 % in differentiated RBCs. Flow cytometry data showed no differences in RBC maturation and enucleation between the wildtype and KO cells. Small blood clots were made using these cultured RBCs and fibrin was successfully visualized and quantified. In addition, lysis of these clots occurred in the presence of tPA.

Conclusion(s): Highly efficient KO RBCs were obtained using CRISPR/Cas9. No differences were observed in the growth and differentiation between wildtype and KO RBCs, suggesting that Cas9-mediated genome editing does not affect the RBC differentiation process. Our data show that these cultured RBCs can be used in assays to study clotting and fibrinolysis.