The organism / system in which you want to edit it would be another important information.

cheers

Hi Francesco, thanks for the reply. It will be within an avian (chicken) system!

Hi J,

In vivo or in-vitro? That would make difference.

I haven't experience on chicken system (we mainly work in mouse in vivo).

In my experience anyway, in cell system to insert large piece of DNA by Cas9 editing it is better to use donour DNA with a selectable marker (i.e. neomycin resistence or whatever)the following use of selective media will hekp to isolate right colonies/cells; this can't help in-vivo editing in embryos for example. In our hand inserting large construct (>200bp) in a knock-in approach is generally unefficient in mouse embryos editing, even if it is feasible.

Some condideration:

.in in Yoshimi et al., 2016 ,Cas9 is expressed together with GFP, that is a TRANSIENT effect, but you wants to TAG a protein (constitutive), if I well understood.

.That's the reason why you need a large donours than "Use single stranded oligodeoxynucleotides (ssODN) like you said".

Or maybe I misunderstood your question.

.Finally; targeting with nicking Cas9 it will be less efficient than the WT Cas9, iven if more safe in terms of off-targeting.

cheers

F.

Hey Francesco,

This is incredibly helpful advice, thank you so much!

I was planning on validating the system works for my target protein in vitro. I will definitely take your advice regarding large donors and cas9 over ncas9. It's good to know re. the 200bp, however I have had no success in finding a tag even near this size!

Just for information, (and it's probably a very obvious answer, sorry!) but why is a large donor needed for constitutive expression?

Thank you so much again,

Jess