hey,
im designing an experiment to optimise prime and base editing epeg/sg RNAs based on their efficiency and off target effects. I understand that using a cell line easy to transfect such as HEKs are ideal for this, and then select the best rna and use in your target cell. But I will need to insert the desired mutations in the HEK cells first… I thought doing the reverse prime/base editing was a bit of a long procedure just to correct the mutation again, so wanted to transduce them with a lenti carrying the gene with the mutation. This gene however does not fit into a lentivirus since it’s quite big, would inserting just a region of the gene that contains the mutations along homology arms (of1kb or how long?) be enough to validate genomic edit? ofc I wouldn’t be able to validate anything at the protein level but just to assess the efficiency at the gene level. has anyone done this or know of a publication which has? Or any reference that might suggest it’s a valid experiment? What do you think? thanks!!!
While your plan sound feasible, I'm not sure it is worth your time. Unless the cells you plan to work in eventually are a real nightmare to work with I would just transfect a plate with each pegRNA / sgRNA, screen the pool by sequencing, and proceed with the one that has the highest percentage of correctly edited genome copies.
There is no point. Why introduce cell type and that vector situation as new variables? This is not going to tell you anything about what happens in your final intended system. Optimize for what you want, as directly as possible.