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Is a crRNA pool more likely to cause off-target effects?

We do not observe a negative effect on cell viability when a pool of four crRNAs is used to target genes for knockout, and increased cellular toxicity is a highly prevalent side effect of off-targeting. We also do not observe viability effects from causing multiple DNA breaks at the targeted gene. However, any guide RNA has the potential to cause sequence-specific off-targets. These off-target effects can be additive when using a pool of crRNAs and potentially lead to false positive or false negative effect on the phenotype. Therefore, follow up with individual crRNAs is highly recommended during hit validation to provide confirmation of the phenotype with several independent reagents.