Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Roesel, KB.
Knock-out of AML-related genes via induction of STOP codons through CRISPR/Cas9 Base Editing.
[ Diplomarbeit/Master Thesis (UNI) ] University of Graz; 2021. pp.45.
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Reinisch Andreas
- Zebisch Armin
- Altmetrics:
- Abstract:
- CRISPR/Cas9 is a genome-editing tool commonly used for gene knockout. Usually, error-prone non-homologous end joining (NHEJ) of Cas9-induced DNA double strand breaks (DSB) is employed for formation of insertions and deletions (indels). Unfortunately, frequent DSBs lead to apoptosis of fragile primary cells, such as hematopoietic stem and progenitor cells. Base editing, a new CRISPR-based application, allows for modification of single nucleotides in the DNA while avoiding DSB. Base editing can be used to introduce in-frame STOP codons via C to T transitions, thereby leading to gene knockout and mimicking of loss-of-function (LOF) mutations. The aim of this project was to employ base editing to knockout genes commonly affected by somatic LOF mutations in acute myeloid leukemia (AML) such as TP53, RUNX1, STAG2 and SMC1A. First, we chose two guide RNAs targeting different loci of the above-mentioned AML-related genes, respectively. We generated the necessary plasmids, followed by an optimization step to gain maximum editing efficiency at maximum cell viability. We could show introduction of in-frame STOP codons in TP53, RUNX1, STAG2 and SMC1A and were able to generate isogenic single-cell-derived clones. While in STAG2 and SMC1A a maximum C to T editing of 50% was achieved, we were able to generate isogenic single-cell-derived clones of TP53 and RUNX1 showing 100% C to T editing. Knockout of the respective protein was confirmed via Western blot. In summary we show that base editing is a powerful tool for gene knockout via introduction of in-frame STOP codons. Our findings will serve as a basis for the implementation of base editing in primary HSPCs, in order to study the gradual accumulation of AML-related mutations.