OC 69.3 - Homology Directed Repair Edited Megakaryocytes For Rapid Screening of Platelet Gene Variant Functions

Background: Functional studies are urgently needed for the mechanistic and clinical interpretation of the numerous variants found by association studies and genetic testing, but are largely missing for variants affecting platelets. The paucity of functional variant validation has been in part because of the inability to make genetic modifications in anucleate cells. We previously published a robust approach to delete and survey platelet gene functions in megakaryocytes called CRIMSON (CRISPR-edited megakaryocytes for rapid screening of platelet gene functions). Here we describe CRIMSON HD (CRIMSON with Homology Directed Repair (HDR)) for the rapid generation and functional testing of precise point mutations, insertions, or deletions in primary megakaryocytes.

Aims: Generate and functionally test genetic variants in human CD34+ cell derived megakaryocytes using HDR.

Methods: Human cord blood CD34+ cells were transfected with CRISPR/CAS9 targeting exon 5 of ITGA2B, and a homologous DNA donor harboring a 6 nucleotide insertion. After differentiation into megakaryocytes, Sanger sequencing was used to validate insertion. Megakaryocyte αIIb surface expression and agonist induced ligand binding were measured by flow cytometry.

Results: We tested CRISPR/CAS9 directed HDR for CD34+ derived megakaryocytes using a homologous DNA donor harboring a two amino acid in frame insertion (Figure 1A-B) described to cause loss of αIIb function without affecting surface expression. As shown in Figure 1C, DNA sequence analysis indicated almost complete (96%) inclusion of the insertion in the genome of HDR edited megakaryocytes. Consistent with this, whereas CRISPR/CAS9 cutting without HDR resulted >90% loss of CD41 and CD61 surface expression, inclusion of R192T193 rescued their expression (Figure 2). Despite rescue of expression, MKs with the R192T193 insertion lost the ability to bind PAC-1 or fibrinogen after agonist stimulation.

Conclusion(s): CRIMSON HD can be used to rapidly generate and functionally test platelet associated genetic variants in their native cellular and DNA context for both mechanistic insight and clinical interpretation.