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CRISPR-Cas genome engineering explained from A to T : Understanding CRISPR genome engineering via a rainbow human embryonic stem cell reporter line to identify pacemaker cells and a MEF2c construct production

Sterckel, Sem A. (2021) CRISPR-Cas genome engineering explained from A to T : Understanding CRISPR genome engineering via a rainbow human embryonic stem cell reporter line to identify pacemaker cells and a MEF2c construct production.

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Abstract:The CRISPR/Cas research domain is a fast expanding domain with lots of potential to open up many doors in other research fields. Using a guideRNA (gRNA) and a Cas9 endonuclease precise double stranded breaks (DSB) can be made in the DNA of cells. The repair mechanism, homology directed repair (HDR), is able to then insert sequences with a size of multiple kilobases (kb). In this thesis a HDR vector targeting the AAVS1 gene for the insert of a MEF2C DNA binding construct with the ability to promote transcription of a mRuby sequence was produced with the use of a sticky end PCR technique. Also a human embryonic stem cell (hESC) dual reporter line, expressing mCherry from the COUP-TFII gene and GFP from the NKX2.5 gene, was targeted for the fusion of the fluorescent protein mTAGBFP with the transcription factor SHOX2 via precise genome editing. With SHOX2 being a perfect reporter for functional pacemaker cells. The MEF2C detecting construct will in future experiments be used to discover underlying mechanisms of MEF2C related diseases like cardiac hypertrophy. And the cells targeted with the mTAGBFP insert are to be made into a stable rainbow reporter line allowing for research in biological pacemakers.
Item Type:Essay (Bachelor)
Faculty:TNW: Science and Technology
Subject:42 biology
Programme:Biomedical Technology BSc (56226)
Link to this item:http://purl.utwente.nl/essays/87665
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