Protein trigger in TALEN, ZFN, ...

[Andreas "Mega" Stuermer]

Hi everyone! 

I have a conceptual question for Prime-Editing and Zinkfinger Nucleases. 

Let's say ZFN:

1) You take a natural protein domain that binds DNA of a specific sequence. 

2) You add a Nuclease domain to the above metnione protein.  

Why doesn't the Nuclease Domain always cut any DNA when it bumps into it, and just when the DNA-binding domain binds DNA? (Unless it's FokI which needs to dimerize if I recall correctly). Is there a term that I can google to find papers? 

I assume there is a conformational change that activates the nuclease domain. But how do you design this? 

Same with CRISPR that changes C->T (I think they termed that CRISPR base editing). You add a deaminase to CRISPR, why doesn't the deanimase just float around in the nucleus and deanimate every base it hops by? 

Any insight would be greatly appreciated! 

[Bryan Jones]

That's a good question. I believe part of the answer is just the ratio of time the protein is bound to the target sequence vs near other regions of DNA. If >99% of the time the protein is bound to the target sequence and less than 1% of the time it's not, then at least 99% of the cutting or base-editing will be on-target. I'm not sure what the dwell time or kon/koff rates are for these proteins, but that might allow you to figure out how much of the time it's bound to the target. Interestingly, similar approaches with transposases haven't really worked. Fusing transposases to TALE domains, zinc-fingers, or Cas9 gives a little enriched transposition at the target, but mostly the transposase still integrates transposons all over the genome. Here's one study that did this: https://elifesciences.org/articles/53868 

--Bryan Jones

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