Design and evaluation of the CRISPR-Cas9 genome editing approach in hippocampal neurons for defining neurodegenerative mechanisms in Parkinson's disease
Gnjatovic, Jelena (2021-08-04)
Design and evaluation of the CRISPR-Cas9 genome editing approach in hippocampal neurons for defining neurodegenerative mechanisms in Parkinson's disease
Gnjatovic, Jelena
(04.08.2021)
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
suljettu
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2021091345920
https://urn.fi/URN:NBN:fi-fe2021091345920
Tiivistelmä
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. In order to improve diagnostic and therapeutic approaches for PD, disease etiology needs to be better understood. Advances in gene editing technologies over the past decade have opened possibilities in translational medicine research. The first aim of this work was to design and generate the transgenic constructs for the CRISPR-Cas9 genome editing. The second aim of this work was to evaluate the gene editing efficiency in hippocampal neurons using immunostaining.
The transgenic constructs in the pORANGE cloning system for CRISPR-Cas9 gene editing in neurons were generated using the traditional cloning approach with PCR-based methods and restriction endonucleases. The resulting constructs were transfected into primary hippocampal neuron cultures and antibody-labelled in immunofluorescence immunocytochemistry. The stained neuronal samples were visualized using confocal and widefield fluorescence microscopy to evaluate the genomic edits.
A total of 20 gene editing constructs were generated in the pORANGE cloning system and confirmed in next-generation sequencing. Gene editing efficiency in the correct orientation was evaluated as the EGFP knock-in reporting either expression or knock-out of the endogenous proteins. The achieved efficiencies for knock-out gene edits in hippocampal neurons were 9%, 30%, and 75%, for each of the three target proteins respectively, while the knock-in mutations reporting the endogenous protein expression need further optimization. These results will support further optimization of CRISPR-Cas9 gene editing both in vitro and in vivo to study the etiology of neurodegeneration in PD.
The transgenic constructs in the pORANGE cloning system for CRISPR-Cas9 gene editing in neurons were generated using the traditional cloning approach with PCR-based methods and restriction endonucleases. The resulting constructs were transfected into primary hippocampal neuron cultures and antibody-labelled in immunofluorescence immunocytochemistry. The stained neuronal samples were visualized using confocal and widefield fluorescence microscopy to evaluate the genomic edits.
A total of 20 gene editing constructs were generated in the pORANGE cloning system and confirmed in next-generation sequencing. Gene editing efficiency in the correct orientation was evaluated as the EGFP knock-in reporting either expression or knock-out of the endogenous proteins. The achieved efficiencies for knock-out gene edits in hippocampal neurons were 9%, 30%, and 75%, for each of the three target proteins respectively, while the knock-in mutations reporting the endogenous protein expression need further optimization. These results will support further optimization of CRISPR-Cas9 gene editing both in vitro and in vivo to study the etiology of neurodegeneration in PD.