Hello,
I currently setting up an experiment to study the role of a set of genes that span 150 Kilobases in my cell line. So I plan on generating a 150 KB deletion by nucleofecting Cas9 with 2 sgRNAs into my cell line. What is the best PCR genotyping strategy to validate my deletion? I was planning on doing three separate PCR reactions (attached in the figure, primers sets are indicated by different colors).
For anyone that has experience doing this, is this strategy okay or are there better PCR strategies to validate a deletion of this size?
Thank you, I really appreciate all the help.
Those primer sets look good and will pick up the deletion but would it be useful to have another primer set either entirely inside the deletion or from outside the deletion into the deletion to show the presence of the normal sequence in case your cell population still has some unmodified cells present from the original cell line?
Remember, you can only find what you are looking for. Paul is correct that you also need include a primer set inside the deletion area and primer set with one inside and one outside. Ideally, you could sequence the "boundaries" of the deletion and design a new primer that will only make a product when the deletion is present.
The primer sets look good. You can also try using the 'green' forward and 'blue' reverse primers for deletion detection.
For Paul Rutland suggestion about ' to have another primer set either entirely inside the deletion ', you might end up with a 'false' conclusion--> the fragment is deleted, but some of the deleted fragments can still be floating around in cells and picked up by PCR. This was reported in site-specific recombinase (SSR)-mediated deletion. The deleted DNA fragments became circular form and floating in cells. The difference is the fragment is linear from CRISPR-mediated deletion. It is more useful to use one primer inside and one primer outside for PCR checking, as mentioned.
Deepak Saini One more thing, I saw your green and blue pairs of primers are very close to deletions site. In a few cases, CRISPR-mediated deletions can rip through a larger fragment than you bargain for. In that case, it can remove/delete your primer priming sites. You should bear that in mind.
I agree with the previous comments that you need at least several primer pairs for the "internal" regions of your deletion. Ideally they should evenly distributed within the region that is being deleted. This is especially important if the cells that you are dealing with are diploid, in which case you could have homozygosity for wild type, heterozygosity, or homozygosity for the deletion at this locus from the CRISPR.
One more small technical thing to consider. For the primers in the green and blue pairs, you have positive controls to show that they are working. What I mean by that is you can use the primers with the WT DNA and they should give you bands; if they don't, you know there might be something wrong with the primers. However, you do not have that for the yellow primers. They won't be able for amplify with the WT DNA because the amplicon is too big. I think if you design the primers carefully, you probably do not need the yellow pair. Instead, you can just use the blue and green primers that are outside of deletion and facing each other for that "spanning" PCR. If you do want to keep the yellow primers, maybe you can test them out by pairing them with the blue and green primers that are inside the deletion to confirm that the primers are good individually at least.
Hope it helps. Good luck!
Yuan-Yeu Yau has raised an excellent point about being cautious with your experiment. You should fully sequence any potentially interesting deletion alleles to make sure that you know exactly what has been deleted.
That will save you a LOT of frustration if your "wild type" primers green & blue give unexpected results.
Deepak Saini Are you only trying 1 sgRNA at one site? I read quite a few CRISPR papers--many tried at least 2 sgRNA for experiment and to see which sgRNA is more effective. The one you choose (sgRNA 1 or sgRNA 2) might not work or with low efficiency. You need to know this too.
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