Simply put, orthogonal validation uses additional methods that provide very different selectivity to the primary method to confirm or refute a finding. All methods are independent approaches that can answer the same question and are used to evaluate and verify the findings. According to Clarence Mills, R&D senior scientist, Horizon Discovery, a PerkinElmer company, the use of multiple methods to achieve a phenotype greatly reduces the likelihood that the observed phenotype resulted from a technical artifact or an indirect effect, thereby increasing the confidence of the results.
“Ideally, the orthogonal method should alleviate any potential concerns about the intrinsic limitations of the primary methodology,” says Mills. For example, if full gene knockout could result in compensatory expression, gene knockdown technologies such as RNA interference (RNAi) or CRISPR interference (CRISPRi) could be used to decrease rather than silence target gene expression, Mills explains. If double-stranded DNA breaks (DSBs) are a concern, alternate technologies that suppress gene expression without introducing DSBs such as RNAi, CRISPRi, or base editing could be employed to validate the result. The methods do not have to be performed in parallel; an orthogonal method is used to subsequently validate genes or loci identified with the primary method.
Examples of orthogonal validation
“One publication that stands out and really demonstrates the power of orthogonal validation is a 2016 paper from the Broad institute,” says Mills.
In this Cell Systems publication1, the authors performed both shRNA and CRISPR knockout screens to identify and validate a set of genes essential for the proliferation of cancer cell lines exhibiting β-catenin activity. They then utilized proteomic profiling and CRISPR-based genetic interaction mapping to further interrogate their candidate genes. This approach allowed the authors to characterize genes that would have been classified as lower confidence hits had they only performed a screen with a single loss-of-function technology and phenotypic readout. Using an orthogonal experimental framework, the team identified new regulators of β-catenin signaling and defined functional networks required for the survival of β-catenin active cancers.
Furthermore, says Mills, with the widespread adoption of CRISPR technologies, orthogonal validation is becoming more prevalent, especially in high impact journals. Another strong application of orthogonal validation that Mills mentions is a 2021 Cell Reports publication2 on the innate immune response to SARs-CoV-2 infection. In this study the researchers used RNAi to screen 16 putative sensors involved in SARS-CoV-2 infection and subsequently used CRISPR knockout to corroborate their results. The findings provided critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.
Many genome editing methods to choose from
Mills highlights a few genome-editing technologies that can be of value in orthogonal validation. CRISPRmod is a newer approach and refers to CRISPR technologies that make use of an endonuclease-dead Cas enzyme, typically fused to a transcriptional effector, to alter target gene expression rather than edit a genomic site. CRISPR activation (CRISPRa) enables upregulation of endogenous gene expression and can be a powerful orthogonal tool to traditional overexpression methods that rely on exogenous expression of a cDNA or ORF (open reading frame) construct. While CRISPRi makes it possible to repress transcription of a target gene, it can be used to validate results obtained with other loss-of-function methodologies such as RNAi or CRISPR knockout.
The emerging technology of base editing allows a researcher to make precise changes to a genomic sequence without introducing double-strand DNA breaks. Like other loss-of-function technologies, it can be employed for orthogonal validation of gene knockdown or knockout. Mills emphasizes that base editing can also increase the granularity of a gene knockout study as it can be used to determine which regions of a gene or genetic element or even amino acids in a protein are critical to function or ligand binding.
“Let the nature of your study guide your choice of methodologies,” advises Mills. “Careful consideration of the function and context of your genes of interest, time point of analysis, and method of reagent delivery, is critical to its success. The use of an orthogonal method not well-suited to the experiment could introduce complexity and uncertainty, whereas a well-designed orthogonal experiment conducted with the appropriate gene editing or gene modulation reagents will enhance the study.”
- Rosenbluh J, et al., Genetic and Proteomic Interrogation of Lower Confidence Candidate Genes Reveals Signaling Networks in β-Catenin-Active Cancers. Cell Syst. 2016 September 28; 3(3): 302–316.e4. doi:10.1016/j.cels.2016.09.001
- Yin X, et al., MDA5 Governs the Innate Immune Response to SARS-CoV-2 in Lung Epithelial Cells. Cell Reports 34, 108628, 2021 January 12. doi:10.1016/j.celrep.2020.108628