P2A-GFP tagging of a weak silent gene

[Dario Besusso]

Hi Everyone,

I am trying to use w.t. Cas9 to tag at the c-term a silent gene using a donor carrying HA(no stop)-P2A-GFP-lx-PGK-PURO-lx-HA. I have now few questions:

1. Do I necessarily need to remove the PURO cassette using Cre?
2. I found unusually tricky to design a screening PCR that won't amplify random insertion (one primer on donor and one on endogenous seq). Do you have any suggestion on how to optimize? Should I use double amount of endogenous primer to compensate for binding to non recombined alleles?
3. If the signal results to be to weak for my applications, is there any way to amplify? How about double the GFP? -P2A-GFP-P2A-GFP?

Any suggestions is very welcome.

Best,

Dario

[Jonathan Geisinger]

1) You should remove the Puro cassette as it seems your strategy totally disrupts the endogenous 3'UTR of your gene of interest.

2) If it's "unusually tricky", your homology arms are probably too big and you're using Taq.  Use Phusion or Q5 instead.  If that isn't an option, you have no real choice except Southern blotting. (There are other recommendations, but those are more expensive.)
3 )Use Clover instead.  The rabbit and goat antibodies for GFP also recognize Clover and it is much brighter than GFP.  mNeonGreen is also a possibility, but you have to purchase a different antibody and the protein itself is from a totally different organism.  

[Brian Lin]

I agree with Jonathan's suggestions. One thing to add, your arms don't need to be as massive as some papers have shown in the way past, when they were 4+ kb in length.

Also, your idea of daisy chaining fluors separated by 2A sequences does work. (Paper...coming...sometime) We have knocked in a doublet construct of Gene-T2A-TdTomato-E2A-TdTomato, except we codon optimized the first TdTomato to minimize the possibility of recombination. Without the codon optimization, it was nigh impossible to see anything but a recombinant mess. Granted, this may be because we used TdTomato, itself a repeat. I would only do a doublet like this if your gene is super low abundance, as it just gives you a headache. If Clover isn't bright enough, a single TdTomato is very bright, as an alternative, and many RFP/mCherry abs recognize that.

Good luck

Brian

[A.N.]

Hi Brian,

Interesting approach that I am also planning to use. I was wondering why you choose the T2A and E2A, and not P2A. And did you add a Furin cleavage signal before the 2A sequences to avoid effects from the tag sequence? Furthermore, my supervisor was suggesting to add a WPRE sequence, but I am wondering whether this would be necessary (I probably try both with and without WPRE).

BR,

Anna

[Brian Lin]

Hi Anna, to be completely honest, we started this project so long ago that the plos one comparison paper had not even been published. We then tested the cleavage efficiency in-vitro and found there was enough product stoichiometry that we didn't go back. If I recall, we got almost 95% efficiency through both 2A peptides as we got nearly double the amount of TD than the original protein.

We did use the GSG linker, without the furin cleavage sequence, as we wanted to use a specific antibody to blot for verification. In hind-sight, we now know that the tag does not affect the function of the original protein.

We did not use the WPRE sequence, as this was purely a knock-in and we did not want to disturb the actual function of the gene. Depending on your application, the WPRE may help greatly.

Hopefully this was helpful, let me know if I can help further,

Cheers,

Brian

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Brian

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[A.N.]

Thanks Brian. As I also want to study the actual function of the gene, I probably also will not need the WPRE than.

Best wishes,

Anna