| Description |
The ETV6 gene encodes a protein transcription factor (TF) that represses gene activity. Mutant ETV6 function is linked to predisposition to leukemia, lower platelet counts, and altered platelet defects, but the mechanisms are unclear. Since the ETV6 TF is a repressor, we hypothesized that pathogenic ETV6 mutations in megakaryocytes (MK), the precursor cell of platelets, would cause overexpression of ETV6 responsive genes involved in platelet function and hematological disease. In this study, we generated pathogenic ETV6 mutations in primary human MKs that will be used in future studies to test the effects of ETV6 mutations on MK development and function. Platelets are anucleate and cannot be directly edited. Instead, CRIMSON (CRIspr edited MKs for rapid Screening of platelet gene functiONs) edits primary human CD34+ cell derived MKs. CRIMSON uses the CRISPR Cas-9 system, coupled with single stranded DNA (ssDNA) donors and inhibitors of nonhomologous end joining to promote homologous directed repair (HDR) in MKs. This system depends on efficient CRISPR cutting to incorporate a single strand of DNA harboring the mutation of interest into the host cell chromosome. Literature review identified several pathogenic mutations that affect platelets including R339C, R418G, P214L, and R396Q. Multiple CRISPR guide RNAs were designed to target these mutations, and transfected into MKs to test efficiency (data quantified and analyzed with sanger sequencing). Transfections were successful at all 4 sites, but cutting efficiencies were low, except for 2 guides targeting the R339C mutation, which had a cutting efficiency up to 83.8%. Next, the 2 guides and different ssDNA homologous donors harboring the R399C mutation were tested for HDR efficiency, and we identified a ssDNA/guide combination that yielded efficient generation of the R399C mutation in MKs. |
