I'm doing CRISPRi with dead spCas9 on mouse cells, and normally we do the following for gRNA design:
1. 20 nt spacer
2. Extra G on the 5' to enhance expression from hU6 promoter.
Final product: 5'-G-spacer-3'
But I accidentally included two Gs instead of 1 G.
Final product: 5'-GG-spacer-3'
Does this matter? I did *a lot* of cloning and cell line generation this way, so I don't know if I will have time to go back to redo it before my contract is up.
Dear Gen, several protocols mention that adding a double GG before the space sequence can increase the expression of the gRNA (even more than a single G), I've been using this for my gRNA designs. You should be fine.
Best,
Diogo
for instance: https://www.nature.com/articles/s41467-017-01836-2
or: https://www.nature.com/articles/s41589-019-0338-y