Base editing with Cas9 and ssODN: gRNA cutting score vs distance

I am designing a CRISPR experiment to correct a point mutation in iPSCs.

I will use a plasmid encoding gRNA, Cas9, GFP/Puro and a ssODN as repair template.

Regarding the gRNA I don´t have lot of options: one cuts very close to the insertion site (2 bp) but has medium on/off target score while the others have higher on/off target score but further away from the insertion site (15-20 bp).

In your personal experience what would you prioritize in such a case? gRNA score or distance from insertion site?

Any comment and suggestion is welcomed.


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Sienna Wyvernalmost 2 years ago

It's always tricky answering this type of question because what you choose to prioritize will really depend on the end-goal of your particular experiment. How much editing efficiency are you looking for? How important is it that you minimize off target or bystander editing for your goal? Here's a link to a recent paper that came out of David Liu's group:

(Open-Access via webpage): https://drive.google.com/file/d/1StERSMpO3h22FglSb9JTdk7p6c86dtAp/view

And an associated mini-site that helps users navigate choosing existing CBEs or ABEs based on what matters most to them (screenshot below). It may be a useful starting point (or at least a helpful reference for the future!).

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