Should I be worried about false positives or off-target effects when screening using the Guide-it CRISPR Genome-Wide sgRNA Library System?
Should I be worried about false positives or off-target effects when screening using the Guide-it CRISPR Genome-Wide sgRNA Library System?
False positives from a screen can result from a guide RNA targeting an off-target sequence that is unrelated to the sequence for which it was designed. We have significantly negated the risk of this happening with the design of the library and the optimization of the screening process. This library contains sgRNAs from the Brunello library which were chosen using algorithms that maximize on-target specificity and activity while minimizing off-target activity (Doench et al. 2016).
Additionally, each gene is targeted by four highly active sgRNAs. We recommend choosing positives from your screen based on the criteria that at least three guides for a given gene must be over/underrepresented after screening compared to their numbers before screening. The probability that three guides for the same gene also target the same unrelated off-target gene is close to zero.
Lastly, false positives can arise from co-transduction events where one sgRNA not related to the knockout phenotype is present in the same cell as an sgRNA that is responsible for that cell's enrichment. Statistically, the ineffective sgRNA will be represented by only one guide, and therefore we can regard it as not being a qualified hit. We can easily limit this phenomenon by adjusting the MOI for transduction to achieve ~30–40% transduction efficiency.
References:Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR Cas9. Nat. Biotechnol. 34, 184–191 (2016).