I want to study the effect of several different mutations on the gene of my interest. But the problem is that there is a very high activity of this protein due to its high endogenous level in HEK293 cells. The assay format I am using to measure the activity of the protein does not allow me to simply work with the overexpression system, as the background activity is too high to detect any fold-change increase. Therefore, I decided to CRISPR-Cas9 knock out (KO) the endogenous gene using pX330 CRISPR plasmid, and then re-express the gene vs. the mutants transiently to rescue the activity.
For CRSPR/Cas9 KO, I co-transfected the HEK293 cells using Lipofectamine2000 with two pX330 plasmids each containing a different gRNA, one targeting the 5’ UTR and another targeting the endoplasmic reticulum-localizing signal sequence of the gene, which is expected to result in a ~400 kB fragment cut out. Puromycin treatment allowed me to select for transfected cells. Later on, I cultured these cells without puromycin. I have not yet confirmed by PCR if the gene fragment is chucked out by CRISPR/Cas9. However, fortunately enough, using my cellular activity assay, I tested and confirmed that the activity of my target protein vanished.
Now, in order to rescue the activity of the protein by re-expression, I would transfect the KO cells with a plasmid carrying the gene which was knocked out. However, if the Cas9 plasmid is still around in the cells, will it target my plasmid which carried the replacement for the KO gene? The 5’ UTR targeting gRNA sequence is missing in my plasmid but the signal sequence is there in my plasmid. Would the gRNA target this on my plasmid and kill my gene of interest not allowing me to re-express the intact protein?
My KO cells still seem to be puromycin resistant suggesting that the plasmid is still hanging around in the cells. Is it possible that Cas9 with the designed gRNAs has integrated in the genome of HEK293? Is it important to check for the Cas9 presence in the KO cells before trying the rescue experiment? There seems to be a 3X-Flag tag on Cas9 in the pX330 allowing to probe for Cas9 on a western blot or I can also check this by PCR amplification of Cas9 gene. Should I make attempts to somehow throw-out the Cas9 of my KO cells such as by culturing the cells for long periods without puromycin?
I am reluctant to make silent mutations disenabling the target of the gRNA in the plasmid which I would use to re-express, because I have designed hundreds of mutants which I should also test, meaning I will have to mutate the same string in all my constructs. Recently, the CRISPR squad has also found out ways to turn off Cas9, should I think in that direction?
Thanks in advance for your helpful responses.
Short answer: Introduce synonymous mutations in the original targeting site (optimally the PAM sequence) in order to prevent a potential recut by the integrated Cas9-sgRNA. For the future: Better select using fluorophores (e.g. using FACS) since antibiotics "enforce" the cell to integrate your plasmid (here the Cas9-sgRNA plasmid). Also it's not good at all the have a potential active nucleaes swimming around in your cell which might off-targets with unpredictable impact. Even if you have no predicted off-targets it's just a matter of time that Cas9-sgRNA is cutting also sequences with 5 mismatches as example.
Dear Dong-Jiunn,
Thanks a lot for your short and clear answer. I have been thinking on the same lines, and I have already ordered the pU6-(BbsI)_CBh-Cas9-T2A-mCherry construct for selection with mCherry. I believe that this construct is deposited to Addgene by your group. This I understood will allow either selection with Puromycin or with mCherry and one can follow the Cas9 clearance with mCherry.
Since, I was not 100% sure if mutating the PAM ensures disabling the gRNA target in my rescue construct, so I designed synonymous mutations rather in the gRNA target sequence which change the sequences by 20-25%, which actually would be around 4-5 mismatches. I think this would work for transient transfections, but I guess as you said it could be lethal if I want to establish a stable line with the rescue construct in the KO background.
And in another lines which we have made using lentivirus transfections followed by puromycin selection, I assume now that there I can forget to use this strategy of synonymously changing the gRNA target instead of PAM, as the Cas9 will hang around there forever. So I should rather mutate the PAM in each rescue construct.
We were also thinking to transfect a tet-inducible gRNA against Cas9 for rapid clearance of Cas9, however this will make things further complicated.
It would be interesting to design something which deactivates the Cas9 immediately after the gene disruption.
Thanks again, your suggestions are really helpful.
simply remove the puromycin, grow single clones, analyze them and then rescue appropriate ones.
If the cells can be transfected, do transfection for the cells with gRNA and Cas9 and then do selection to kill the non-transfected cells( puromycin for 2 days). Then do limited dilution or single cell sort to get individual colonies and screen for the KO. The chance of both gRNA and Cas9 integrate into genomic DNA is low , but transient trasfection is good enough or Cas9 and gRNA to cut the genomic DNA. So you can just put wild type gene back to restore the gene expression.
Rescuing the activity of a gene knocked out by CRISPR/Cas9 involves a few strategic approaches. Here's a structured guide to understanding and potentially reversing the effects of a CRISPR/Cas9-induced knockout:
It's important to note that the feasibility and efficiency of these strategies can vary significantly depending on the gene in question, the organism, and the specific mutation introduced by CRISPR/Cas9. A carefully planned and executed experimental approach is crucial for the successful rescue of gene activity.
Perhaps this protocol list can give us more information to help solve the problem.
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