Why is the expression level of Cas9 important?
Why is the expression level of Cas9 important?
When expressed at high levels, Cas9 nuclease can be toxic to mammalian cells, but expression must still be high enough for efficient gene editing. It is, therefore, well worth putting in some effort up front to create the best Cas9+ cell population.
To determine the optimal Cas9 expression for your screening cell line, transduce target cells with different amounts (MOI) of Cas9 lentivirus, select each population on puromycin, and then monitor the cells lines’ growth rates (see figure below). Choose a Cas9 population transduced with the highest MOI that does not exhibit any change in cell growth compared to cells that do not express Cas9.
Expand the population to make a master cell bank with enough vials for all your future screens.
Cas9 affects growth rate at high expression levels in some target cells. In the figure above, samples of HT-29, 293T, and A375 cells were transduced using low (blue), medium (red), and high (green) MOI, respectively. Two of the cell lines demonstrated a slower growth rate at the highest MOI while the third cell line was tolerant of all MOIs tested.