Is the knock-out permanent if I transient transfect gRNA into a stable Cas9-expressed cell line?
I think it will be permanent knock-out because once the indel is created, it is irreversible regardless of gRNA's expression. Then the parent cells will divide into daughter cells with this indel. Am I right?
Thanks!
Except for cells that can be inherited, all sgRNAs gradually disappear.
you will have a population of cells that descend from your successfully transfected cells. the size of that population will depend on your transfection efficiency. that population of descendants will have the knockout permanently, as Zhanqi says the gRNA will disappear since it is transiently transfected and you are not performing any selection.
Once you have made alterations to the genomic DNA using CRISPR, all daughter cells will inherit that change. Since you're making a knockout with CRISPR it will involve NHEJ, and once that process has taken place, all daughter cells will inherit the altered genomic DNA.
You will probably want some way to select for the knockouts, or isolate single cells to establish clones since otherwise your population after transfection will be a mixture of cells that have the knockout and cells that don't.
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