Mastering screening: Validating and following up on hits from loss-of-function screens.

The advent of genomic screening with CRISPR-Cas9 has sparked a revolution in our understanding of biological processes, cellular behavior, drug interactions, and development. Whether using pooled, highly multiplexed lentiviral sgRNAs or arrayed synthetic crRNAs, the essence of screening is to ask specific questions with tens of thousands of gene-specific reagents to pinpoint which genes are involved in a process. The most common method is CRISPR knockout (CRISPRko) screens, with CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) also gaining traction^1,2.

Running a screen can be logistically challenging, but the real work begins afterward: validating which hits are genuine and informative.

Here we describe some of the most used strategies for hit validation.

Deconvolution

Deconvolution is about breaking down something complex into its basic parts. In a primary screen, multiple reagents targeting the same gene are often used together. To confirm a hit, you can test each reagent individually in a secondary screen. For instance, mixing four to six crRNAs targeting the same gene into one well can be cost-effective. If this mix shows a hit, you can evaluate the quality by seeing how well each individual reagent replicates the result. This process of deconvolution, or separating the effects of each individual reagent, allows for a more accurate validation of hits.

Orthogonal reagents

Using orthogonal reagents—those that affect the same gene but through different mechanisms—is a common way to validate hits. For instance, in CRISPRko screens, RNAi reagents could verify if a phenotype caused by DNA-level editing can also be seen with mRNA-level silencing of the same gene. This approach provides a robust validation of hits by confirming the observed phenotype through multiple independent methods³.

Knockout cell lines

CRISPR-Cas9 has made creating knockout cell lines faster and cheaper compared to older technologies like ZFNs and TALENs⁴. Now, knockout cell lines are readily available as catalog items, saving time and effort. Using a characterized isogenic line allows for more complex experiments (like paired knockouts or synthetic lethality) and provides a stable background for screens. A haploid line ensures complete knockout of a gene, creating a clean model. As a validation tool, a knockout cell line can be used for rescue experiments by reintroducing the gene’s function with a cDNA or ORF. It also enables matrixed knockouts to study gene modulation in the context of a gene’s knockout.

Looking for a partner to work with on hit validation to free up in-house resources? Find out how our preclinical services team can help.