University of Twente Student Theses
CRISPR Activation via nucleofection on hESCs, hESCs derived cardiomyocytes and practical applications during differentiation
Alvis Arango, J.F (2025) CRISPR Activation via nucleofection on hESCs, hESCs derived cardiomyocytes and practical applications during differentiation.
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| Abstract: | CRISPR Activation (CRISPRa) is an innovative technology derived from the CRISPR/Cas9 system, enabling the targeted and reversible upregulation of gene expression without introducing permanent changes to the genome. By utilizing a catalytically inactive Cas9 (dCas9) fused with transcriptional activation domains, CRISPRa directs enhanced expression of specific genes, offering a powerful tool for investigating gene function, studying developmental pathways, and exploring possible therapeutic targets. Its versatility makes it particularly valuable in contexts where precise temporal and spatial control of gene activation is required, such as stem cell research and disease modeling. In this study, a CRISPRa protocol was implemented in human embryonic stem cells (hESCs) to activate the cardiac-specific genes NR2F2 and MEF2C. This protocol was then optimized before moving onto cardiomyocytes. A nucleofection protocol had to be devised for hESC-derived cardiomyocytes, addressing the unique challenges of transfecting differentiated cells, which include lower proliferation rates and reduced viability. Finally, CRISPRa was used during the differentiation of hESCs into cardiomyocytes to investigate its potential influence on differentiation. The results demonstrated successful activation of target genes in hESCs, with qPCR showing an increase in expression for both targeted genes. The nucleofection in cardiomyocytes proved challenging at first, however after implementing multiple strategies like special medium cocktail and different nucleofection programs, acceptable transfection efficiency and confluency post transfection was achieved. Lastly, CRISPRa was performed during differentiation, where cells managed to survive the process and developed some cardiac markers like Troponin T (cTnT). This work serves as a foundation for further protocols involving CRISPRa in hESCs and nucleofection in cardiomyocytes for potential cardiac research. It also provides preliminary insights into the practical application of CRISPRa during key processes like differentiation. Finally, various avenues for optimization are discussed as well as current roadblocks and potential obstacles. |
| Item Type: | Essay (Master) |
| Faculty: | TNW: Science and Technology |
| Subject: | 44 medicine |
| Programme: | Biomedical Engineering MSc (66226) |
| Link to this item: | https://purl.utwente.nl/essays/105065 |
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