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CRISPR/Cas9 induced overexpression of GABA receptor α1-subunit and CB1 receptor

Ryde, Martin (2017) MOBT01 20162
Degree Projects in Molecular Biology
Abstract
Epilepsy is one of the most common neurological diseases that affects 1 % of the human population. After an initial insult, the brain undergoes several changes that lead to hyperexcitability. These changes include downregulation of both GABAA receptor subunits and CB1 receptors. These receptors normally work to decrease excitability, so their downregulation leads to an increase in excitability. If this downregulation could be prevented or counteracted, the severity of the epilepsy could be reduced. We tried to do this by utilizing the CRISPR/Cas9 system; this system makes use of guide RNAs that, in our case, are designed to target the CNR1 and GABRA1 genes, which code for CB1 receptors and the alpha1 subunit of GABAA receptors,... (More)
Epilepsy is one of the most common neurological diseases that affects 1 % of the human population. After an initial insult, the brain undergoes several changes that lead to hyperexcitability. These changes include downregulation of both GABAA receptor subunits and CB1 receptors. These receptors normally work to decrease excitability, so their downregulation leads to an increase in excitability. If this downregulation could be prevented or counteracted, the severity of the epilepsy could be reduced. We tried to do this by utilizing the CRISPR/Cas9 system; this system makes use of guide RNAs that, in our case, are designed to target the CNR1 and GABRA1 genes, which code for CB1 receptors and the alpha1 subunit of GABAA receptors, respectively. Here, it will activate these genes and increase their expression. In this study, we successfully upregulated CNR1 and GABRA1 expression in primary neurons using lentiviruses with CRISPR/Cas9. This shows great potential for future experiments and will be the basis for in vivo experiments. (Less)
Popular Abstract
Receptor overexpression by CRISPR/Cas9

Epilepsy is one of the most common neurological diseases, affecting 1 % of the human population. It is characterized by recurrent epileptic seizures. The process by which epilepsy is developed is called epileptogenesis. During this process a lot of changes occur in the nervous system that lead to imbalances between the excitatory and the inhibitory networks in the brain. This leads to hyperexcitability. In this study we wanted to use the CRISPR/Cas9 system to upregulate GABAA subunit alpha (GABRA1) and Cannabinoid receptor 1 (CB1) in hopes of decreasing the hyperexcitability.

In order to do this we made use of the CRISPR/Cas9 system, which can target specific sequences with the help of guide... (More)
Receptor overexpression by CRISPR/Cas9

Epilepsy is one of the most common neurological diseases, affecting 1 % of the human population. It is characterized by recurrent epileptic seizures. The process by which epilepsy is developed is called epileptogenesis. During this process a lot of changes occur in the nervous system that lead to imbalances between the excitatory and the inhibitory networks in the brain. This leads to hyperexcitability. In this study we wanted to use the CRISPR/Cas9 system to upregulate GABAA subunit alpha (GABRA1) and Cannabinoid receptor 1 (CB1) in hopes of decreasing the hyperexcitability.

In order to do this we made use of the CRISPR/Cas9 system, which can target specific sequences with the help of guide RNAs (gRNAs). Here it would cut the sequence, but the Cas9 has been modified to activate the sequence instead. So we made viruses that target the GABRA1 and CNR1 genes, and induces overexpression. Cells were then infected with the virus. Next RNA and protein were extracted from the cells. The RNA was used to make cDNA and then to run a real time PCR, that will show how much of our target RNA there was in the cells. The proteins were used to run western blot to see if the protein is there.

We first tried to overexpress CNR1 in neuroblastoma cells (N2A cells), but this did not work due to these kind of cells being heavily modified. So we decided to try out the viruses in mouse fibroblast cells (MEF). Here we could see a four-fold increase in relative expression compared to a control virus. Then when we had seen that the overexpression actually worked, we proceeded with our desired target, primary neurons. Here too we saw a four-fold increase in relative expression.

Next we tried to overexpress GABRA1 in primary neurons. Here we could only see a 50 % increase in relative expression. What we had measured so far was the increase in mRNA in the cells, but an increase in mRNA does not necessarily mean an increase in proteins. In the case of CB1, there was such a low concentration of CB1 so that we could not detect any protein with our antibody. In the case of GABAA, we could see a 39 % upregulation.

Taken together this shows great potential for future in vivo studies, which are currently underway. Hopefully this technique will be useful in treating epilepsy that the drugs that we have now can not.

Master’s Degree Project in Molecular Biology, Medicinal Biology, 60 credits, 2016-2017
Department of Biology, Lund University

Advisor: Marco Ledri
Epilepsy Centre, Department of Clinical Sciences (Less)
Please use this url to cite or link to this publication:
author
Ryde, Martin
supervisor
organization
course
MOBT01 20162
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8926751
date added to LUP
2017-10-03 11:20:44
date last changed
2017-10-03 11:20:44
@misc{8926751,
  abstract     = {Epilepsy is one of the most common neurological diseases that affects 1 % of the human population. After an initial insult, the brain undergoes several changes that lead to hyperexcitability. These changes include downregulation of both GABAA receptor subunits and CB1 receptors. These receptors normally work to decrease excitability, so their downregulation leads to an increase in excitability. If this downregulation could be prevented or counteracted, the severity of the epilepsy could be reduced. We tried to do this by utilizing the CRISPR/Cas9 system; this system makes use of guide RNAs that, in our case, are designed to target the CNR1 and GABRA1 genes, which code for CB1 receptors and the alpha1 subunit of GABAA receptors, respectively. Here, it will activate these genes and increase their expression. In this study, we successfully upregulated CNR1 and GABRA1 expression in primary neurons using lentiviruses with CRISPR/Cas9. This shows great potential for future experiments and will be the basis for in vivo experiments.},
  author       = {Ryde, Martin},
  language     = {eng},
  note         = {Student Paper},
  title        = {CRISPR/Cas9 induced overexpression of GABA receptor α1-subunit and CB1 receptor},
  year         = {2017},
}