Establishing gene function, whether in healthy development or disease, lies at the heart of genetic research. Once we discover what a gene does, we can start to understand disease mechanisms and potentially identify novel therapeutic targets.
But as with all scientific investigation, the strength of our conclusions depends on the rigor of our experimental design. So, how can we build greater confidence in our findings? Let’s take a closer look.
The role of loss-of-function studies
Loss-of-function (LOF) studies involve investigating the effects of reducing or eliminating the activity of a gene or protein. These studies have broad applications, including:
- Understanding biological processes
- Identifying disease mechanisms
- Developing therapeutic strategies
- Validating potential drug targets
Researchers can achieve LOF using a variety of tools. These include gene editing applications such as CRISPR-Cas9 for gene knockout, CRISPR interference, RNA interference (RNAi) methods like siRNA and shRNA, as well as antisense oligonucleotides that can bind to mRNA and prevent translation. The effects of these perturbations are then analyzed to determine the gene’s role in cellular processes, phenotypes, or disease development.
While each of these techniques offers valuable insights, no single method is perfect.
Challenges of loss-of-function studies
A major challenge in LOF studies is distinguishing true effects from artifacts. False positives can arise when a gene or protein is incorrectly identified as essential. These errors can occur due to various factors, including off-target effects affecting other genes or unintended consequences of the experimental set up. False negatives, on the other hand, can occur when a gene or protein’s role is missed. Contributing factors include functional redundancy masking the effect of a gene knockout, insufficient knockdown efficiency, techniques not sensitive enough to detect subtle phenotypic changes, or compensatory mechanisms that activate following gene disruption.
To minimize these risks, careful experimental design and validation are essential. This can be achieved by using appropriate controls, verifying the efficiency of gene knockdown or knockout, and considering more than one method to interrogate gene function.
Why orthogonal approaches matter
The use of multiple LOF technologies to target the same gene is a powerful approach to reduce ambiguity. If two different technologies, such as CRISPR-Cas9 for gene knockout and shRNA for gene knockdown, produce similar results, it is more likely that the phenotype observed is due to the gene of interest and not off-target effects.
CRISPR-Cas9 targets a genomic locus to introduce a short insertion or deletion (indel) mutation, causing a frame shift mutation that results in a non-functional protein. In contrast, shRNA works by targeting a mature mRNA transcript and results in active degradation or translational repression of the mRNA transcript. Because the two technologies operate through different mechanisms, any overlap in phenotypes adds a layer of validation that a single method can’t provide.
Case study: Cardiac differentiation research
A recent study demonstrated the power of this approach. Revvity scientists investigating cardiomyocyte differentiation used both CRISPR knockout and shRNA expression to investigate the roles of key transcription factors in the early stages of cardiomyocyte differentiation from induced pluripotent stem cells (iPSCs).
CRISPR knockout of these genes resulted in a clear reduction in the number of cells that undergo complete differentiation to cardiomyocytes. When the same genes were targeted with shRNAs, similar results were observed validating the initial findings. This study is the perfect demonstration of how combining orthogonal LOF approaches adds rigor to experiments. It helps confirm that the observed effects are indeed real and gene-specific, especially when working with cardiac tissue that is in limited supply and technically challenging to work with.
For a deeper look at how this dual approach was used to explore cardiac differentiation, including the full methods and results, read our full application note: CRISPR knockout and shRNA: Orthogonal tools to perturb gene expression in iPSC-cardiomyocyte differentiation.
