So I deleted an exon of a gene in a cell line using CRISPR. The region deleted is around 1500bp. I isolated colonies, extracted DNA and ran a PCR with primers designed upstream and downstream of my guideRNAs. So without that region gone, I'd have an amplicon of ~1600, but with the deletion I'd have around ~300 bp amplicon.
I took DNA from what I thought were deleted samples and non-edited cells, and used a different set of primers that were designed to lie within the region I deleted. I was expecting to get no amplification in the deleted samples and an amplicon of 800bp in the non-edited samples, but I ended up getting amplification in all samples present. One of my deleted samples had a noticeably fainter band than the others still.
I'm trying to figure out what the issue was. Could homologous recombination have occurred? Though why would the first PCR indicate a probable deletion? I'm going to do sequencing as well.
Have you thought about using multiplex ligation-dependent probe amplification (MLPA)?
Article Detecting exon deletions and duplications of DMD gene using ...
In case of occurrence an HDR repair, you should have seen the same band in the first gap-PCR step! Are you sure that in the first stage, you have extracted the DNA sample properly and added to all of the PCR microtubes?
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