Between June and August 2024, our Master’s students Siv, Eystein, and Janaarthan successfully defended their Master’s theses. 👏 🥳
We are incredibly proud of their achievements and honoured to have worked alongside them at Haapaniemi Lab. They excelled throughout the year and performed exceptionally during their defences. We also extend our heartfelt thanks to the opponents and examiners for their valuable contributions to these successes.
You can read more about their projects below.
Siv Skundberg Jensen
- Master thesis title: Correction of a Signal Transducer and Activator of Transcription 1
(STAT1) gain-of-function mutation in T cells by applying the CRISPR-Cas9 technique - Date and place of defense: 18 th June 2024, OsloMet
- Main Supervisor: Shiva Dahal-Koirala
- Co-supervisor: Emma Maria Haapaniemi
This thesis aimed to correct the STAT1 gain-of-function mutation in a patient’s T cells in vitro, using the CRISPR-Cas9 technique, to improve cell functionality. Patient-derived T cells were transfected by electroporation with Cas9, guide RNA, and a repair template designed to correct the STAT1 mutation. The best-performing guide RNA and repair template were identified by screening available options.
The level of mutation correction, achieved through homology-directed repair, was quantified using droplet digital PCR. To assess the impact of mutation correction on T cell functionality, phosphorylated STAT1 levels were analyzed by flow cytometry. A reduction in phosphorylated STAT1 levels indicated successful mutation correction. We achieved up to 30% gene editing and observed a corresponding decrease in phosphorylated STAT1 levels in the corrected T cells, compared to uncorrected T cells. These findings suggest that CRISPR-Cas9-mediated correction of the STAT1 gain-of-function mutation in vitro leads to a promising improvement in the functionality of the patient’s T cells.
Eystein Lie Asdal
- Master thesis title: Optimization of CRISPR-mediated gene knock-in in monocytic THP-1
- Date and place of defence: 27th June 2024, NMBU
- Main Supervisor: Anna Zofia Komisarczuk and Carolina Wieczorek Ervik
- Co-supervisor: Emma Maria Haapaniemi
In this project, we used a Cas9-gRNA complex and a single-stranded DNA template to introduce a silent mutation in the ADA2 gene in THP-1 cells, a cell line commonly used in immunology research. We optimized culture conditions, electroporation settings, and buffers to achieve a homology-directed repair (HDR) efficiency of up to 42%, which is the highest reported in this cell line. Additionally, we tested various non-homologous end-joining (NHEJ) inhibitors and observed that specific inhibitors reduced both HDR and NHEJ repair.
However, the use of a commercial molecule to enhance HDR led to a significant increase in HDR efficiency, reaching 56% only after 10 days. These results provide valuable insights for improving CRISPR-mediated gene editing in THP-1 cells.
Janaarthan Geneshan
- Master thesis title: epegRNA Screening Platform for Primary Immunodeficiency Mutations in HEK293 cells and Hematopoietic Stem and Progenitory Cells (HSPCs)
- Date and place of defence: 19th August 2024, NTNU
- Main Supervisor: Anna Zofia Komisarczuk
- Co-supervisor: Emma Maria Haapaniemi
In this study, we discuss the setup of the engineered prime editing (epegRNA) screening
pipeline in HEK293 cells to edit single nucleotide mutations causing primary immunodeficiency diseases (PPID). We evaluate efficiency and design of single epegRNA and establish the efficient protocol for testing epegRNAs utilizing lentivirus system.
We select the best epegRNA guides that will be tested and optimized in Hematopoietic Stem and Progenitory Cells (HSPCs) in the virus-free system, preparing grounds for therapeutical use of prime editing in immunodeficient patients.