I have validated the guideRNAs in hela but when I am trying to edit stem cells using the same guideRNAs there is cut seen even in my untransfected samples. I have used different guideRNAs but both my UT samples are showing editing similar to that of my test sample. Is it possible the genes are mutated during passaging and culturing. Or can it be due to pcr polymerase being used while amplification.
hi
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709030
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500303/
Thanks Noor. I went through both the articles but was not able to find the reason why I am seeing an edit happening in my un-transfected samples when I am performing the T7 assay.
Hi Thulaj,
Could there be a SNP somewhere in the region that you are amplifying? If so, it would induce a "cut" by T7 assay. You can probably find out by sequencing your PCR product.
Hi George, I had thought of the same thing some time back but the possibility of all my different gRNAs towards different genes having a SNP and exactly at the same location where they have been designed should be very rare.