I have purchased a lentiviral CRISPR library. However I wish to replace the marker gene in the library backbone with one of my own. I'm trying to decide what the best strategy to do this is:
1) Pick one colony of the library prep, clone in the new marker gene, then use this as the backbone to clone back in the sgRNA library.
2) Make a large library prep with the original library, and then do a large-scale cut and paste back in of my new marker gene. This would involve running the whole library on a gel and I have concerns about efficiency of the process, but would be a lot less steps.
Any practical advice/ resources I should check out please?
I have not done what you're trying to, but from a purely theoretical perspective I don't see how option 2 would work. Option 1 will work, you'll just have to re-clone all of your gRNAs into the backbone, basically creating a brand new library (which there are protocols for).
I am curious however if you do try option 2, if it would work.
Good luck!
Hi, in theory, both methods can work, but I agree that option 1 is better, but make sure that there is no carryover of the "library" portion when you replace the selection marker, which is not an easy task to verify. A better course of action would be to obtain the original backbone (which should be available) and replace your selectable marker and then go for the transfer. Another critical aspect is how you will "transfer" your library back and ensure that you have as little empty vector as possible (golden gate, gibson, etc etc) which will depend on the library itself and that you have no vector carryover.
The bigger problem is library bias/shift/loss of representation, regardless of which way you proceed. This is because by the time you re-make your library, it will have gone through 3 or 4 (and possibly more) amplification steps since its original creation by synthesis (combination of bacterial amplificaiton and/or PCR), and each amplification skews the library in various ways. Unfortunately, the "best" way would be to re-synthesize and clone into your new backbone, but that requires a more substantial investment up front. That being said, it is what it is, and the important step is to determine your library bias and verify that it is acceptable, and this will require a higher depth of sequencing.
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