Dear Ms. Lima,

Your question actually tackles unsolved problems about gene dominance and gene dosage in polyploids.

CRISPR/Cas9 is a method outside of my comfort zone, but am I right in assuming you'd select your successful knock-ins based on somekind of selection with antibiotics? Such selectable marker genes are often dominant and it may well be that you have positive lines with only one out of three of your knocked-in alleles.

If your knock-ins create a phenotype (e.g. gene expression) proportional to the copies present, you could identify lines with one, two or three copies of your knock-in. Such lines are in themselves very interesting if you aim at answering questions regarding gene dosage.

There's a body of theory about gene dosage balance, that may help you.

Cheers

Marc

Hello Marc,

Thanks for your answer. Yeah, I select my cells with antibiotics, but then both the transfected pools and isolated clones are sequenced to check the editing efficiency. I guess when doing so it will possible to evaluate how many copies has been edited as well.

Thank you very much!