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CRISPR/Cas9 gene-editing tools construction

Sagerud, Michael Venbakken; Strand, Sebastian Rishaug
Bachelor thesis
Published version
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URI
https://hdl.handle.net/11250/2999706
Date
2022
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  • TKD - Department of Mechanical, Electronics and Chemical Engineering [131]
Abstract
Type 1-diabetes is a disease where the body destroys its own insulin-producing, pancreatic beta cells. The pancreatic

alpha cells produce the blood sugar-elevating hormone glucagon from the precursor protein proglucagon.

Marking the proglucagon gene with CRISPR/Cas9 mediated knock-in of green fluorescent protein (GFP) might allow for

visualising the dynamics within the cell under differentiation from stem cell to somatic cell.

To perform the preferred knock-in, the gene editing tools were constructed. A combined vector of single guide RNA,

Cas9 nuclease and a reporter for the Cas9 nuclease was constructed. A donor DNA template vector consisting of the

GFP between sequences homologous to each side of the Cas9 cut site was cloned with In-Fusion multi-fragment

cloning. Sanger sequencing confirmed that both vectors were successfully cloned before the combined vector was

successfully transfected into human embryonic kidney cells. T7 Endonuclease I assay estimated an indel occurrence of

14,4% for the combined vector.

The combined vector showed that it did perform cutting, and future research will reveal if the constructed donor DNA

template vector results in a knock-in of GFP in the proglucagon gene.
Publisher
OsloMet-Storbyuniversitetet

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