Hello everyone
I have been doing CRISPR to knockout a gene from my HEK-293 cell line. I have transfect with SgRNA-cas9 and now my cells have been growing for a week. I am planning on doing DNA isolation and sanger seq for my gene of interest. my question is how can I be sure that I had heterozygous or homozygous mutation ? should I do qpcr? do you recommend other ways than sanger seq to confirm the knock out?
Thanks
Just do a regular PCR across your target site. If there are two bands of different sizes it is a heterozygous knockout.
If you used CRISPR you are most likely to have a deletion, especially if you used a double gRNA method.
Sanger sequencing will tell you the exact base(s) that were changed. It will be a mixture of all alleles that are present.
Unless you already have a developed assay with robust controls, qPCR is not the way to go.
You'll see peaks corresponding to the mix of all bases present at each site for all alleles.
It's possible to see small InDels and SNPs in Sanger, as long as both alleles are in equal proportion.
If you want to get really particular, you could do PCR, then clone the PCR products & sequence ~8 colonies. That would show you the exact change in the target region.
Just to clarify on the prior answer to just use regular PCR and look for band sizes. This approach has lots of limitations. For one, you can only resolve a 20% size difference with PCR. So, if you see two different bands, that means you do have 2 size differences. However, one could be WT and the other mutant. Or, both could be mutant. You'll need to sequence to know for sure exactly what you got.
As someone who does CRISPR work, I highly suggest cloning and sequencing.
The bulk Sanger sequencing will give you nice clean single peaks for WT or a matched set of mutations (exact same change in each allele). If the alleles are different at all you'll see mixed peaks.
Well, the short answer is that you can see a frameshift INDEL if the change alters your PCR product length by at least 20%. Otherwise, it will run at the same size as your WT band.
Are your colonies independently regenerated after CRISPR treatment? The number to screen will depend on the efficiency of the targeting rate.
Also, Hek293 cells are a bit genetically complex. Is your target gene diploid or in one of the triploid chromosomes?
As a note, you can't really know if what you are getting/doing is a frameshift mutation. The DNA repair after Cas9 cutting is stochastic so you can get a variety of mutation types. Typically, repair after CRISPR-Cas9 editing gives deletions, many of which can lead to frameshifts.
Dear Danny, you should integrate your PCR fragments (inserts) to plasmids, then transform competent E. Coli bacteria with those plasmids, containing your insert. We used Zymo Mix&Go competent cells and pGEM-t-easy plasmids. Only one PCR fragment could integrate to one plasmid and one E.coli cell picks up only one plasmid. Thus, in case of diploid cells, your two different PCR fragments (from the two alleles) integrate to different plasmids, with 50-50 distribution. That's called PCR fragment cloning. Check our article with the protocol:
Article The Creation of a Multiallele Knockout Genotype in Rabbit Us...
In case of a regular diploid cell, sequencing 6-8 bacteria colonies would be enough.
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