Primary Menu

Education, Events, Publication

Funding & Recognition

Generation of Point Mutants in the ETV6 Gene in Primary Human Megakaryocytes

Year: 2023

Presenter Name: Puja Batchu

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 functional platelet defects. We generate pathogenic ETV6 mutations in megakaryocytes (MK), the precursor cell of platelets, to test mutation effects on ETV6 function and MK development. Since the ETV6 TF is a repressor, we predict that mutations will cause overexpression of ETV6 responsive genes involved in platelet function and hematological disease.
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 incorporates a single strand of DNA harboring the mutation of interest. Literature review identified mutations that affect platelets including R339C, R418G, P214L, and R396Q. Multiple CRISPR guide RNAs and ssDNA donors were made using a CRISPR HDR Design Tool that computes the number of base pairs to cut site and predicted accuracy. Each guide was 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. Genetically edited MKs will be used to create platelets that will be tested for RNA expression changes, and changes in platelet function. This will provide insights on how ETV6 mutations affect platelet function in hematological diseases.
University / Institution: University of Utah
Type: Poster
Format: In Person
Presentation #D6
SESSION D (3:30-5:00PM)
Area of Research: Health & Medicine
Faculty Mentor: Jesse Rowley