Are the guide RNAs evenly represented in your library? Does this change when producing and transducing virus?

In a perfect whole genome library, each guide RNA is present in an equal copy number (equal representation). In practice, no library is perfect, due to variability in the processes used to make it (oligo synthesis, cloning, transformation, library amplification, DNA extraction, etc.). Our experience of creating, amplifying, and validating libraries ensures that our library is produced to high [...]

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 [...]

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, [...]

How many total transduced cells do I need for a whole genome sgRNA-library screen and why?

We recommend performing each screen using a minimum of 7.65 x 107 cells transduced at 40% efficiency when using the Guide-it CRISPR Genome-Wide sgRNA Library System. See the calculations below: Total number of guides:19,114 genes targeted by 4 sgRNAs each = 76,456 sgRNAs (plus 156 control sgRNAs) = 76,612 sgRNAs Number of cells transduced per guide:At a low sgRNA representation in the transduced [...]

Which cells should I use for genome-wide sgRNA screen?

The cells need to be a good surrogate for your experimental system but easy to grow and transduce. Primary cells might be the goal but can be difficult to scale up because they often do not proliferate or require elaborate culture conditions. We suggest substituting with a related transformed line for the primary screen, followed by use of more relevant primary cells for follow-up confirmation [...]