you may want to try to see the difference in signal intensities of encoded products by Western (half in hetero- and almost nothing in homo- vs intact control). It works for many non spliced genes

You have multiple options actually

1. Perform TA cloning

2. Sanger sequencing and observe the electropherogram

3. Perform deep sequencing on the PCR product, generated using primers near to your mutation area. Take care of the PCR product length.

All these methods can be used, although I prefer Method 3, since it is fast and also gives you a definitive allelic frequency. Although TA cloning can also provide the same result.

Good luck!!!

Hi Dekel,

Sushree gives very good suggestions - personally I cut the DNA fragment out of an agarose gel, purify it and then send it for sequencing in the first instance. If the electropherogram you get from this is nice and clean, then you can assume that the sequence is homozygous. However, it is isn't a clean sequence, I would recommend TA cloning (we use the pGEM-T vector from Promega), because that allows individual PCR fragments (i.e. individual alleles) to be cloned into a vector backbone. After ligation and bacterial transformation, the plasmids can then be screened and sequenced so that you can read each allele separately.

One thing just to mention though - be careful on how far away from the target site you are designing your PCR primers for this kind of analysis. If your primers are close to your genomic target site and the CRISPR-Cas9 has caused a large deletion in one allele, but homology directed repair successfully occurred in the second allele, your PCR primer binding sites may not be present in the allele with the large deletion. This may explain why you are only seeing a single DNA band via agarose gel electrophoresis. I have seen deletions of >50 bp in some instances in my mutagenesis experiments with CRISPR-Cas9, so would advise you design two or three pairs of PCR primers at different distances from your target site (e.g. up to 100-150 bp each side of the target site). This will give you more chance of catching any large deletions/insertions.

Best wishes,

Jonathan

Thanks for the answers! Sadly, I was not notified that I had answers to my question and never checked here.

What I ended up doing was Tetra-Primer ARMS-PCR, which indicated heterzygosity even though the previous sequencing and gel analysis indicated homozygosity.