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Another key aspect to consider when planning CRISPR screens or custom cell line editing experiments is to determine if the target gene‐of‐interest (GOI) is expressed in the host cell and whether it is essential for cellular viability. Online tools are available and can be used (Chen et al. 2017) to help determine this. However, essentiality for some genes is context dependent (Wang et al. 2015) and as such caution on data interpretation is advised (Kimberland et al. 2018). Regardless, highly amplified genes in cancer cell lines appear to be unsuitable for gene KO studies, since simultaneous on‐target cleavage of a high number of alleles may affect cell viability. This has been shown to result in false positives/negatives in library screens where cell viability was used as an enrichment step (Aguirre et al. 2016; Munoz et al. 2016). One approach to address the challenge of essentiality for CRISPR screens is to use CRISPRi, which relies on dCas9 that does not alter the sequence of genomic DNA. However, CRISPRi may not be suitable for all targets, as it can downregulate multiple genes at bidirectional promoters (Rosenbluh et al. 2017).

To separate technical artifacts from true hits and to obtain a deeper understanding of how the target(s) of interest function, one can adopt a dual screening strategy, where two complementary screens (e.g. CRISPRi and CRISPRa) are performed in parallel (Jost et al. 2017; le Sage et al. 2020). Combining screening platforms substantially augments the quality and value of data derived from the screening campaigns, as well as providing novel insights not accessible when using one technology alone. For example, whereas CRISPRko will reveal targets where complete protein removal is required for a phenotype to emerge, CRISPRi could be used to investigate levels of repression needed to elicit a phenotypic response.