Actually there is a particular reason why I want to do something like this.
So I am trying to insert in C.elegans, using CRISPR, a human ortholog of a gene. I’m using the approach published by D. Dickson. And using his vectors pDD282.
My current rescue construct is Left Arm–Ortholog–SL2–(FP-SEC)–Right Arm.
The problem that I’m having is that I’m unable to get any positive F1s carrying the extrachromal array. And I think the reason for this is that the human Ortholog is lethal in the high concentration.
And the Left Recom Arm actually has an active Promoter that belongs to the C. elegans gene, which would be disrupted after the ortholog is inserted.
So I was thinking to get rid of the let-858 utr sequence that is present after the GFP in pDD282 vector.
My having none or reduced expression of the ortholog as an extrachromsal array. I should be able to inject high concentration of the rescue plasmid (keeping the HR efficiency high).
And once I heat shock to remove the SEC I would get the expression from only the native promoter and utr as a single or double copy.
Of course I can change the around the sequence of the rescue plasmid, by having the ortholog just before the right arm, but I wanted to avoid extra cloning.