I need to KO a gene in a stem cell lines cultured in medium ,However,transect and pick up clones is difficult. So I use Lenticrispr which express lentivirus to KO. under the circumstances, How to put back this gene? because CAS9 is always expressing, It will also cut the put back CDS
You can introduce silent mutations in the gRNA binding site/ PAM so it will not be recognized by your guide anymore.
Yoav Lubelsky Thank you very much. Just to be sure it can'be cut by CAS9, How many bases should be mutated ? Is Just one NGG site enough? I'm worrying that It will still be cut because of mismatch.
Theoretically, mutating the PAM sequence should be enough but if you want to be on the safe side just make all the possible changes that will not affect the protein sequence.
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