gRNA designing for Cas9 Knock In

I hope you all are doing well.

I am going to design gRNA for mpeg.1.1 gene knock In in zebrafish. I have few quires, Please I need your help

1. Is it compulsory to put the GG sequence at the 5' end of gRNA?

2. How can I design gRNA with zero off-target effects?

3. How can i evaluate off-target effects virtually. ?


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Lemon Pegasusover 1 year ago

1. If you are using a U6 promoter, it is necessary to add a single G at the 5' end of gRNA. If you are using a T7 promoter, then it's necessary to add GG at the 5' end for efficient transcription.

2. It depends on where in the genome you want to introduce your edit. There is no 'technique' that will allow you to design a gRNA with absolutely zero off-targets; it just depends on your region of interest within the genome. So, you can use online tools such as CRISPR Optimal Target Finder, Benchling, CcTop, CRISPOR, etc., to help you with the gRNA design and evaluating potential off-targets. If you are lucky, your region of interest may have zero off-targets.

3. Perhaps you mean to ask what would happen as a consequence of off-targets? Well, simply put, your Cas9-gRNA complex would bind somewhere else (the off-target) and introduce the cut (DSB) there.

Spend a fair amount of time finalizing your strategy before starting your experiments.

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