Hello everyone,
I am currently trying to design guideRNA for a CRISPR knockout experiment using the desing-tool CRISPOR.
CRISPOR designs all the oligos you need for the CRISPR experiment.
I just noticed one strange aspect:
When designing the oligo for U6-driven expression in mammalian cells the tool keeps adding a C directly next to the restriction site (i looked up the linked plasmid) and a corresponding G at the 3' end. Note that i am not talking about the G after the restriction site, which is neccessary for U6-driven expression.
Do you have any idea what the added C is good for?
Thank you for your help.
(the bold sequence corresponds to the GOI, the restriction site is supposed to contain 4 residues. one additional C is added in each strand)
Dear Tim,
I would recommend that you check the Addgene page for your plasmid https://www.addgene.org/43860/
and pay attention to how you are supposed to clone your guide into the plasmid
Then actually align your two primers as a double-strand ie sense 5-3' and antisense 3-5'
This should get you your answer.
Guide RNA have 'G' at the 5' end are considered to be more efficient for genome editing. In case the guide RNA site does not have 'G' at the 5' end, an additional 'G' is usually added.
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