Low editing efficiency seen by TIDE analysis

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!

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White Elfover 3 years ago

How does your single guide chromatogram look like? Did you see extensively mixed peaks after the targeting site?

Mauve Impover 3 years ago

the chromatogram using single gRNAs looks completely normal and no mixed picks at or after the expected cleavege site. After reading this paper "Parallel Sequencing Used in Detection of Mosaic Mutations: Comparison With Four Diagnostic DNA Screening Techniques" I was wondering if it is possible that each of the different mutations induced by the single gRNAs are not represented enough and I am losing them in Sanger?

We will move to NGS next to verify either the absence of mutations when treated with single gRNAs (which to me is very strange) or the presence of different mutations each with a low efficiency.

White Elfover 3 years ago

It does sound very strange. Maybe you shall do T7E1 assay to double check as well.

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