I used Crispr/Cas9 technology for gene knockout in Hek293 cells. I screened cell clones for absence of target protein by western blotting. When I send negative clones for sequencing there was no mutation in the target region. Why is that?
It is possible that one of the alleles of the target gene has not been nicked by Cas9 and so you are sequencing a pool of DNA (presumably from a PCR around the target region) that contains a mix of the WT and the mutant alleles. If the mutant allele has been amplified at a comparable rate to the WT, maybe you will see a second trend line, below the peak, in your DNA sequencing chromatograph.
The best way to confirm in such a scenario would be to PCR amplify your gene of interest, T/A clone it into a vector, pick single ligated colonies from a LB plate coated with X-gal and sequence multiple colonies. It reduces the chances that you are sequencing a pool of DNA and you would be looking at bonafide single DNA molecules from the original PCR.
Hi thanks. I in fact sequenced cloned PCR fragments. Maybe my primers are too close to the target site, so that I cannot pick up indels?? I amplified a 115bp fragment just up and downstream of gRNA sequence.
If it was 115bp total, that sounds like a very small region, if it is 230bp total, that sounds like you should be able to see some indels or mutations.
An alternative way to check this would be to use a surveyor nuclease assay. I think you would need a larger gene fragment to do it though.
Thanks very much for your advice. I will try to PCR a larger fragment. Thanks.
- Badges
- Science method