Genomic screening with CRISPR-Cas9 has become a mainstay in furthering understanding of biological processes, cellular behavior, drug interactions, and cellular development. Performed using either pooled, highly multiplexed, lentiviral sgRNAs, or arrayed synthetic crRNAs, the screening process itself can be distilled to the concept of interrogating a specific question with tens or thousands of gene-specific reagents with the intent to determine which genes are specifically involved in the process. The most common approach is CRISPR knockout (CRISPRko) screens, although CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) are rapidly emerging areas. While running a screen can be a daunting logistical process, the most significant work takes place afterwards: determining which hits are real and informative (hit validation). There are several strategies that can be employed for hit validation, here we present and discuss the most commonly used.


Deconvolution, at its most basic level, is the process of resolving something heterogeneous into its basic components. In a primary screen, it is often the case where several reagents targeting the same gene will be used in a mixture, and the strength of the hit can be elucidated in a secondary screen by examining each reagent individually. For example, it is more cost-effective to mix four crRNAs targeting the same gene into a single well and use them as a single reagent. If the phenotype from a crRNA pool is determined to be a hit, the quality of that hit can be evaluated by determining to what degree the individual reagents can recapitulate the outcome. A gene with three of four individual reagents confirming the phenotype might be given precedence in follow-up work over one that only gave one of four.

Orthogonal reagents

Use of orthogonal reagents, those that have a similar effect on a target gene but work through different biological mechanisms, is one of the most common ways to confirm that the phenotype associated with knockout or modulation of a specific gene is indeed a valid hit. In CRISPRko screens, RNAi reagents can be used to verify if a phenotype due to functional knockout from DNA-level editing can be recapitulated with mRNA-level silencing of that same gene.

Knockout cell lines

The emergence of CRISPR-Cas9 technology has significantly reduced the time and costs associated with creating knockout cells lines relative to older technologies such as ZFNs and TALENs. As such, knockout cell lines are now readily available as catalog items, saving a great deal of time and effort. Use of a characterized isogenic line opens the possibilities of more complex experimental paradigms (e.g. paired knockout, synthetic lethality, etc.) and creates a stable background for running screens. When a haploid line is used, thus guaranteeing complete knockout of a gene, a very clean background model can be created. Importantly, as a validation tool, a knockout cell line can be used for rescue experiments by introducing a cDNA or ORF to restore the gene’s function. Furthermore, it opens the possibility of matrixed knockouts where gene modulation in the context of a gene’s knockout can be studied.