Hi!
I am working with HEK293T and CRISPR/Cas9 technology. I have generated some KO clones with a small deletion of our gene of interest (related to coenzyme Q biosynthesis), by plasmid transfection and paired nickases, and it has been incredibly efficient.
I have isolated some clones, and after some time, the frequency of multinucleated giant cells has increased a lot (in some clones more than in others). I have never seen this phenotype before, and I was wondering if someone has, or knows to what it could be related. I don't know if it is something specific of our gene, or if it could be related to any offtargets of the technique.
I would appreciate any answer or comment.
Thank you!
I have no idea about the gene you are working on. i am working on CRISPR/Cas9 and some times some clones with my desired change, for example deletion, don't grow and some of them with the same change grow.
you didnt mention that what mechanism you use to repare DNA after CRISPR/Cas9 cut!
HR? NHEJ? MMEJ? In HR we have more confidence in DNA reparement but NHEJ and MMEJ is a not accurate cell DNA repair system that sometimes can effect genes beside the your desired deleted sequence. you can check it by doing PCR and sequencing to be sure there is no change beside of deletion point. on the other hand deletion is not simple like this and maybe deletion of one part can effect genome long interaction and effect other genes that for checking this item you have to do 4Cseq experiment.
If the gene you are working or beside it there are no house keeping gene , this phenotype could be related to off target function of CRISPR/Cas9.
does your cells look like this one...these are Hela cells genome edited for a new protein of unknown function..stained for tubulin
weird and interesting. do you have a picture
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology