Why do we need multiple guides targeting the same gene?

Even when working with well-designed sgRNAs such as those included in the Brunello library, there is a chance for off-target edits. In the context of a genome-wide knockout screen, off-target edits that affect the phenotype of interest can result in false positives, because cells expressing the sgRNA that produced the off-target edit could become enriched or depleted in the screened population, implying that the intended target of the sgRNA is involved in the phenotype. Given that the likelihood of several different guides generating the same off-target edit is negligible, the inclusion of multiple guides targeting the same gene provides a powerful means for avoiding false positives in a screen. When a researcher observes enrichment or depletion of several different guides targeting the same gene, he or she can be reasonably confident that knockout of the intended target of the sgRNA is responsible for the phenotype rather than an off-target edit.
Following positive or negative screens the confidence is far higher when you can confirm that multiple guides targeted to different sites in the same gene show up as positives in your data analysis.
We recommend further investigation for those genes where 3 or 4 of the guides targeting that gene show as positives in the data analysis, as these are unlikely to be artifacts.

Gene editing products