Strategies to validate loss-of-function gene targets involved in the mesenchymal to epithelial transition

Assessing gene target function is commonly done through loss of function studies. In particular, RNA interference (RNAi), CRISPR-based gene editing (CRISPR knockout) and CRISPR interference (CRISPRi) are used. Each technology has its own particular strengths when applied to attenuate a target gene, but also come with a set of constraints and considerations. Combining orthogonal loss-of-function (LOF) methods in parallel (orthogonal validation) can help eliminate the possibility of false negative or positive results. In this manner, orthogonal validation is an essential component of a well-designed and -controlled experiment.  An additional benefit of synergistic testing is the possibility of revealing additional properties of the target gene due to the unique properties (effect kinetics, degree of target loss, etc.) of a particular LOF methodology.

Employing orthogonal technologies in parallel to disrupt gene function, each with a different mode and action, and application-specific benefits and considerations, provides an effective target validation approach (compared to one methodology alone).

  • Benefits of orthogonal validation include: 
    Identification and elimination of specious results due to experimental artifacts
  • Lowering impact of experimental difficulties inherent to a particular methodology
  • Outcome validation and increased scientific rigor
  • Elevated chances of discovering additional gene functions or properties

Here we present a study where we have employed orthogonal target validation strategies with genes  involved in maintenance of the mesenchymal-to-epithelial transition (MET). The mesenchymal to epithelial (MET) transition is sometimes considered a reverse paradigm for tumor metastasis. The mesenchymal cells are amorphous, unstructured, migratory, and invasive and revert backwards to the epithelial cell type which are well structured, non-invasive, non-migratory and well anchored. This process goes from a metastatic phenotype to a maintained static phenotype. MET also appears to play a central role in metastatic colonization, where mesenchymal tumor cells revert to a more epithelial state to proliferate and form growths at secondary sites.

In this work, three target-directed LOF platforms are assessed at the level of cellular migration and adhesion markers, two phenotypic hallmarks surrounding the MET, and measured in cultured cells.  This multiplatform orthogonal setup was used to validate and compare target gene knockdown and knockout at four key regulators of the MET:  ZEB1, ZEB2, PI3Ka and AKT2.

By using an orthogonal LOF framework, we:

  • Validated three LOF platforms for significantly depleted protein for all methods and gene targets as well as the level of RNA transcript (RNAi and CRISPRi) and gene disruption by indel formation (CRISPR knockout).
  • Found that all orthogonal methods could effectively modulate cellular migration in a mesenchymal cell line, thus validating the technologies on a functional level.
  • Identified that certain adhesion markers exhibit a response specific to the LOF platforms tested, due to either temporal attributes and/or levels of gene target. 

In short, by enabling corroboration between LOF platforms, orthogonal validation generated confidence in these experimental findings. In addition, we found that adopting an orthogonal study framework revealed that some findings might have been otherwise overlooked if using a single LOF technology.

To learn more about this work, check out our orthogonal validation application note for more details.

Download application note

A demonstration of orthogonal validation to study genes associated with metastatic phenotypes. 

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Written by Brian Ziemba, Senior Scientist II, R&D, Revvity
Dr. Ziemba joined the Revvity R&D team in 2021. Brian obtained his PhD in Pharmacology and Structural biology where he explored properties of alcohol- and odorant-binding proteins. Thereafter, he studied signaling arrays that govern chemotaxis as a postdoctoral researcher and managed the biochemistry arm of an integrative physiology of aging laboratory. He is currently fascinated with the field of genome engineering and enjoys family, cats, music and the outdoors.


  1. Yang, J., Antin, P., Berx, G. et al. Guidelines and definitions for research on epithelial–mesenchymal transition. Nat Rev Mol Cell Biol 21, 341–352 (2020).