Hi,
So I've got the bi-allelic sequence of several Knock Out cell lines I've generated. I assume that the mutations have been caused by NHEJ after the DS break by Cas9. There is variation in the mutations, but I expected that from the random nature of NHEJ.
My question is what is the range of mutations that are expected when NHEJ has taken place? How far from the target site would be considered OK to assume the repair had caused a mutation? Would a 120bp deletion be a possibility with NHEJ or are there other mechanisms altering the genome?
I've tried searching for this on pubmed but I keep wading through off-target papers, which isn't really what I'm looking for as the mutations are kind of on-target.
Any papers or other sources exploring this would be greatly appreciated.
Thanks
In our experiments we have seen mutations going from deletions of 5bp to insertions of 3bp. But virtually any mutation size is possible (like 120bp). However, cells with those events are less probable to survive due to the toxicity of such large deletions. And usually the cut happens very close to the target region +/- 3 nucleotides aprox.
Both NHEJ and TMEJ (end-joining by polymerase theta) repair CRISPR/Cas9 induced breaks. Mutational signatures are described here: Article Mutational signatures of non‐homologous and polymerase theta...
See fig 2d, indeed deletions of 120 bp are possible, based on this plot I would say it's more likely that deletions of this size result from TMEJ rather than NHEJ!
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