I have used CRISPR plasmid pnCasBEC to edit a single base in the S. aureus genome and introduce a premature stop codon in my gene of interest. Transformed S. aureus are selected for, gDNA purified, PCR amplified (with Vent), and Sanger sequenced. I can detect the intended base edit (CAG --> TAG) but at that edited base location, there is always another nucleotide peak that is smaller. I don't see this same ambiguity at other nucleotide locations. I even regrew several glycerol stocks of edited bacteria and repeated the procedure to confirm a "confident" CAG-->TAG. I still get some underlying peaks at the edited position, even equal in size now to the T peak on the chromatogram. This makes the sequencing call hard to nail down. Since these mismatches aren't occuring anywhere else in the sequenced PCR product and it's not at the extreme 5' or 3' end of the sequencing read, it makes me think it's not a difficult and/or impure sequencing template and that it's a real, heterogeneous mixture of gDNA sequences.
My question is whether I might be growing a bacterial colony that really is a mixture of gDNA sequences at that nucleotide (some edited to TAG and some remained unedited). There could be pressure to NOT get edited to a premature stop, right? Has anyone every seen this in their data?
