I have generated a 20 bp deletion in an exon of gene X in zebrafish. The mutation is inherited. Screened for x-/- by PCR/ sequencing and crossed the positive fish (F2 generation). Their progeny show the 20 bp mutation in the genomic DNA when checked by standard PCR but shows a clean WT sequence when sequenced the amplicon from the cDNA obtained from the same progeny.
This actually is a very interesting question!!! and I want to say, "yes", it might happen.
I have read somewhere that some deletions get silenced after a few generations (this I read about plants, but I'm not so sure about zebrafish).
Theoretically, there can be no mutations because CRISPR technique is more specialized and more accurate than other technologies
In reality, mutations can occur because experiments on CRISPR are still very few
did you have no template controls in all of your pcrs?
I am thinking pcr contamination of the deletion template makes the fish look affected but different primers for the cDNA do not amplify the contamination so correctly show the normalallele. If the NTCs are negative then this is not the cae
Ajay Deepak Verma It's clear, your progeny is heterozygous, and the clean WT sequence you got is actually coming because you picked up the WT allele.
Thank you all for your suggestions and comments. Niloufar Pirayesh , this phenomenon is known in zebrafish too. I have one mutant line which has del 13 bp mutation at the same target region and that expresses at transcript level too.
Paul Rutland, I have used NTC for all my PCRs.
Salah Adlat, these parents were genotyped by tail clips and also crossed with wild type fish. Their progeny (randomly choosen 10-12 larvae) were separately genotyped and were found hets (which meant the parent fish that was crossed with WT fish is homozygous for deletion). Now if deletion is there in both the alleles, where from WT sequence comes up? Even if these parents are hets then also the pcr product (generated from cDNA from 3dpf embryos) which was sequenced should show noise at the position of deletion in the chromatogram.
Ajay Deepak Verma Let’s say you genotyped the F0 and suppose one of the parent (F0) was homozygous knockout (you have to confirm that whether F0 is het or homo) and you crossed it with WT, the offspring F1 should be at almost 100% hets according to the Mendelian ratio, or 50% WT + 50% het if your F0 was het and crossed with WT. Now the F1 offspring have no homozygous knockout, they are hets so the WT sequence you got is coming up because of the WT allele. Now to get the homozygous knockout fish you have to intercross the het fish and do your experiments on the F2 offspring where you can get a pure homozygous knockout.
@ Salah you are right. That's exactly I have done the experiment. The F3 embryos I have checked and their parents. (F2) were screened to be homozygous. Trouble is in F3 generation(progeny of homo*homo) at cDNA level it looks wt but at DNA level, it's a deletion in the exon.
Article A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disrupt...
This article may help you
Good question indeed, and very curious on why the cDNA looks like WT while on DNA the deletion exists..
consider it like DNA repair because you have played with a short segment. And, there maybe possible false results as well.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes