Guide design

I want to validate some genes of interest coming out from the
screening with the gecko v2 library.
I'd like to take the guides of the library to singular validate in my
target cells.

Normally I design the guide in the following way:
fwd: 5'-CACC- guide sequence- 3'
rev: 3'- AAAC- reverse guide sequence-5'
Then I do the annealing and clone the annealed guide into the vector.

But I was wondering how to design the guides coming from the library
because I can not understand if they are sense or antisense.
If the guide in the excel file recognises the antisense strain (3'-5')
should I consider to design the flanking region in the opposite way?
So it would be:

fwd: 5'-CACC- reverse guide sequence- 3'
rev: 3'- AAAC- guide sequence-5'
where "guide sequence" is the one found in the excell?

Is there a way to understand it or all the guide in the file are
thought to be designed in the "first-normal (sense) way"

Hope you can help,
many thanks for helping!


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Fuchsia Kelpieover 1 year ago

The library file lists the guide sequences, regardless of whether they target sense or antisense strands. You can clone individual guides from the library as you do normally.

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