Maybe someone here can help me understand my recent TIDE results: I am using CRISPR/Cas9 delivered by ribonucleoproteins to target my protein of interest. qPCR of the targeted gene showed a decrease of more than 50% in its levels when using gRNAs in singleplex. Using dCas9 as a control I we saw no effects indicating that our results were not due to transcriptional interference. I have done Sanger sequencing in the mix amplicons after PCR amplification using primers outside of the targeted region (at least 200nt upstream and downstream). I was very surprised to see that the editing efficiency was so low (between 0.5-2% for different gRNAs). When I use a combination of 2 gRNAs I can see that the sanger chromatogram is all over the place right after the predicted cutting sites of the 2 gRNAs so I believe the gRNAs are working as expected. ____
Would you believe these results are expected since I am doing Sanger in mix populations and the very low represented species with the mutations are not seen or is it really that my editing efficiency is so low? Even if there is more than a 50% decrease at the RNA levels observed by qPCR? ____
Sorry for the long explanation!