All-in-one vector systems have two main advantages:
- Cells only need to be transfected once.
- gRNA/Cas9 expression is driven in an ideal 1:1 ratio.
Dual vectors, where Cas9 and gRNA are expressed independently on separate constructs, are more suitable if you plan to express multiple gRNAs for multiplex targeting. For these applications, Cas9 should first be stably expressed in the cell line, after which the cells can be transfected with different gRNA vectors to generate a cell pool.