Why do you recommend a 30–40% transduction efficiency and not higher?

A key goal in producing a Cas9+/sgRNA+ cell population for screening is to balance the need for every guide RNA to be well represented in the screen with having each cell express only a single sgRNA. Transduction efficiencies of 30–40% are expected to yield the highest quantities of cells expressing a single sgRNA while maintaining a reasonable culture size prior to selection in hygromycin (Miles et al. 2016).
This titration is simplified when using the Guide-it Genome-Wide sgRNA Library System due to the presence of the mCherry fluorescent protein on each sgRNA vector (see map). Simply test a small amount of your library virus prep and measure mCherry expression by flow cytometry or microscopy. For your full-scale screen, use the MOI that transduces 30–40% of the cells.
Reference:Miles, L. A. et al. Design, execution, and analysis of pooled in vitro CRISPR/Cas9 screens. The FEBS J. 283, 3170–3180 (2016).

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