Collateral effects of CRISPR
1 Formål
The purpose of these experiments is to investigate unforeseen effects of CRISPR editing of genes involved in puberty. These experiments are parts of the NFR funded project Ecogene (ID 294840). We will CRISPR edit zebrafish embryos and investigate off-target effects and large structural changes around the target site. This can be done in embryos. However, in the last few years it has become clear that CRISPR editing can induce compensational expression of related genes (because of the generation of premature stop codons), and this must be investigated at stages where the genes are normally expressed. This means that we need to let some of the fish grow until puberty to see if related genes can compensate for the knocked out genes. In addition, we aim to use alternative strategies for gene editing that avoid generation of premature stop codons, to see if this might not trigger the compensational expression. Therefore, we will use the new method CRISPR prime editing and CRISPR promoter knock out and let the fish grow to puberty. The expression of all genes in these fish will be measured with RNAseq in relevant tissues (pituitary, brain area controlling pituitary). We also aim to do single cell RNA sequencing to track the changes in expression of single cells in the pituitary and brain.
2 Skadevirkninger
No negative effects of the fish are anticipated, and if we see that our gene editing is causing any suffering, or malformations, we will terminate the experimental animal.
3 Forventet nytteverdi
The results from these experiments might change the way we do CRISPR in the future. Doing traditional knock-out is maybe not the best way of knocking out the function of a gene because of the compensational expression that can take over the function of the gene. These experiments might also unravel the "true" function of genes involved in reproduction which has never been knocked out without compensational expression before.
4 Antall dyr og art
3 genes, 3 treatments, 2 timepoints, 755 fish maximum in total. Species: Zebrafish (Danio rerio)
5 Hvordan etterleve 3R
The gRNA we are using will be tested before the experiment, so that we only use the most efficient one, to avoid raising many fish that are not edited. Co-injection with a gRNA targeting the melanin production pathway will let us pick out embryos that are probably not rightly injected, and will further ensure that we will only raise the successfully edited fish. Additionally, we will do multiple analyses on the same animal at the end-point to maximize results obtained per experimental animal.
The purpose of these experiments is to investigate unforeseen effects of CRISPR editing of genes involved in puberty. These experiments are parts of the NFR funded project Ecogene (ID 294840). We will CRISPR edit zebrafish embryos and investigate off-target effects and large structural changes around the target site. This can be done in embryos. However, in the last few years it has become clear that CRISPR editing can induce compensational expression of related genes (because of the generation of premature stop codons), and this must be investigated at stages where the genes are normally expressed. This means that we need to let some of the fish grow until puberty to see if related genes can compensate for the knocked out genes. In addition, we aim to use alternative strategies for gene editing that avoid generation of premature stop codons, to see if this might not trigger the compensational expression. Therefore, we will use the new method CRISPR prime editing and CRISPR promoter knock out and let the fish grow to puberty. The expression of all genes in these fish will be measured with RNAseq in relevant tissues (pituitary, brain area controlling pituitary). We also aim to do single cell RNA sequencing to track the changes in expression of single cells in the pituitary and brain.
2 Skadevirkninger
No negative effects of the fish are anticipated, and if we see that our gene editing is causing any suffering, or malformations, we will terminate the experimental animal.
3 Forventet nytteverdi
The results from these experiments might change the way we do CRISPR in the future. Doing traditional knock-out is maybe not the best way of knocking out the function of a gene because of the compensational expression that can take over the function of the gene. These experiments might also unravel the "true" function of genes involved in reproduction which has never been knocked out without compensational expression before.
4 Antall dyr og art
3 genes, 3 treatments, 2 timepoints, 755 fish maximum in total. Species: Zebrafish (Danio rerio)
5 Hvordan etterleve 3R
The gRNA we are using will be tested before the experiment, so that we only use the most efficient one, to avoid raising many fish that are not edited. Co-injection with a gRNA targeting the melanin production pathway will let us pick out embryos that are probably not rightly injected, and will further ensure that we will only raise the successfully edited fish. Additionally, we will do multiple analyses on the same animal at the end-point to maximize results obtained per experimental animal.