Are the guide RNAs evenly represented in your library? Does this change when producing and transducing virus?
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 standards and that representation of guides included in our system is as equal as possible.
For the Guide-it CRISPR Genome-Wide sgRNA Library System, the sgRNA content of the library plasmids is verified in every lot by NGS to contain >90% of the sgRNAs within a 10-fold distribution range. We have additionally measured representation in a transduced cell population and confirmed an extremely high correlation between the starting plasmid material and the transduced cell population (Spearman and Pearson correlations >0.95). This high correlation shows that it is possible to maintain representation through all the steps from transfection, harvest of the virus, and transduction of target cells using our format and protocols. The cells produced using this system are then the starting point from which you begin your screening process.