I think you can simply assay for the presence/absence of the protein of interest (for example, by western blotting if you have an antibody against that protein). Of course, you can isolate the modified protein and assay for its activity if it has a known activity. And you can quantify the enzyme's metabolic product if you know that the particular enzyme is solely responsible for that product.

Hi,

As mentioned above, you may simply do a WB to confirm the OE/KO. Additionally, if you have a particular region, where you insert/delete a DNA sequence, you may run PCR on in with set of primers, which fit only to WT/modified sequence (basically, that's how you do genotyping with animals).

Of course, the enzymatic assay would be the most strong confirmation of the change of the activity of the enzyme, though it is not always feasible. You may also try to look for direct downstream targets and do WB on them, showing the change of their expression, for example.

Best,

Michail

Hello,

1) Given that the efficiency of Cas9 is often limited, our preferred method to confirm the presence of subclones carrying the target modification is ultradeep sequencing: basically high-fidelity PCR amplification of the target locus followed by NGS sequencing of the amplicon. Typical coverage is around 10000x. If we find evidence of subclones carrying the target variant we perform subcloning to isolate the mutated line. If, for some reason, you expect a high efficiency then a PCR followed by Sanger sequencing is enough. We don't use ASO-PCR for this task, as often the result of a knock-out Cas9 is not entirely predictable.

2) It's totally related to the specific activity of your protein of interest, so it could be an ELISA assay, a kinase activity assay and so forth. Provided that you isolated a clone (i.e. cell-line) carrying the modified gene/protein you could either perform the specific assay on, say, the total lysate or you could go for an immunoprecipitation to pull-down your target protein and then proceed with the enzymatic test but this, again, depends entirely on the specific protein you're studying.

I do not understand why CRISPR (clustered regularly interspaced short palindromic repeat)-associated (cas) protein, which is only present in bacteria, is present in humans or rat or mouse.

I do not understand why bacterial enzyme (bacterial enzyme has no glycochains) can similarly works in humans.

Human enzyme works mainly under the control of glycochains (please see file; JMBT Alopecia 1).