Is oligo DNA Phosphorylation dispensible for cloning gRNA into px459 plasmid?

[jason.l...@gmail.com]

Hello who'd concern my topic, recently I need to clone gRNA into px459 plasmid. According to the instruction of Feng Zhang's lab, one of step is to phosphorylate the oligo DNA. I wonder whether this step is dispensible or indispensible. and what is the mechanisms? 

Thank you for who could give me the accurate anwers!

[Petr Tomek]

Hi Jason, I am convinced that there is no need to phosphorylate the guide inserts unless you dephosphorylate the vector. I have not used the plasmid px459 but the AIO-GFP plasmid. When I tried to ligate phosphorylated guides into the AIO-GFP using a one-step protocol (combined digestion+ligation), it never worked. Only non-phosphorylated guides worked in this protocol. Based on these observations, I conclude that phosphorylation is not necessary and can actually inhibit proper ligation. The digested vector retains the phospho groups required for the ligation (unless you dephosphorylate it), so there is no logic here supporting the need for phosphorylation of the insert. In my opinion, phospho-insert can actually oligomerise and undergo certain unwanted reactions that messes up the cloning. Hope that helps. Cheers, Petr

Dne pátek 17. března 2017 10:44:21 UTC+13 jason.l...@gmail.com napsal(a):

[Jonathan Geisinger]

That is certainly a conclusion one could make.

To address Jason's question, in using the Zhang lab protocol, yes, the phosphorylation of the oligo DNA is required.  The mechanism is basic molecular cloning. It is better to have your backbone dephosphorylated because it is using carrying your resistance marker and because oligomerization of your insert is usually much less problematic than oligomerization of the backbone.  Because the Zhang lab protocol includes dephosphorylating the backbone, ligation with unphosphorylated oligo DNA would not work as there would be no phosphate groups for the ligase to act on.

[Petr Tomek]

Yes, I completely agree with you Jonathan. However, to my knowledge, and now correct me if I am wrong, the initial Nature protocol for CRISPR/Cas9 genome engineering (Ran et al., 2013) instructs to perform backbone digestion and ligation simultaneously in one tube. I am not aware of any intentional dephosphorylation occurring in this particular reaction.

Jason, it depends on what protocol you are following. If your protocol asks for vector dephosphorylation, then the phosphorylation of your oligos is indispensable as Jonathan says. However, if you are not dephosphorylating your vector backbone, the insert phosphorylation is most likely dispensable as your vector still retains the phosphate groups necessary for the ligation.

Good luck with your cloning and let me know how it worked out. I am curious to know if the phosphorylation can be omitted. Best, Petr

Dne neděle 19. března 2017 17:39:08 UTC+13 Jonathan Geisinger napsal(a):

[Jonathan Geisinger]

The addgene protocol from the Zhang lab (I think Le Cong wrote it, but I'm not sure on that) predates the Ran et al. protocol and used (and still does!) a phosphatase in the digestion reaction.  Maybe the Ran protocol was an early step towards library construction using Gibson assembly.