I am trying to add a florescent tag using CRISPR to a protein at a midpoint (not at the C or N terminus) I am having trouble finding any papers where this has been done before. Can someone please direct me to a paper where this has been done?
Also is there a website or software I could use to predict how adding the florescent tag in different places will affect the protein structure?
Thanks.
It has been done before (see papers cited below). A main question to ask before you decide on a strategy to do so is how much you know about the structure of your target protein. The fluorescent protein has to be inserted in such a way that it does not disrupt the folding path and native structure of your target protein. If you have a structure or reasonably good homology model, you may be able to rationally design your fusion construct, select an insertion point in a broad loop and adapt the linker lengths. If you lack reliable structural information, you may use secondary structure prediction, hydropathic profiles and sequence conservation to find positions that are reasonably likely to tolerate such an insertion. The less information you have on the structure of your target protein, the more you will have to rely on selecting the working solution from a wide variety of different constructs.
Article A new way to rapidly create functional, fluorescent fusion p...
Article High-Throughput Fluorescent Tagging of Full-Length Arabidops...
https://www.frontiersin.org/articles/10.3389/fpls.2013.00214/full
https://academic.oup.com/bfg/article/9/2/129/216264
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