Determining the subcellular localization of a protein within cells is crucial to determining its overall function. Several techniques exist for determining subcellular localization; however these are not matching the requirements of modern molecular science. SLENDR is a novel tool recently developed that allows for rapid protein localization, high-throughput readouts, and is scalable to many endogenous proteins and cell types.
The location of a protein within a cell gives us a great insight into the protein’s role in different cellular processes, and overall function. Therefore, the development of an accurate and effective method for identifying protein subcellular localization is very important, and eagerly sought after by researchers. Commonly used techniques for protein localization exist like immunostaining for endogenous proteins, overexpressing fusion proteins, and protein fluorescence. Unfortunately, these techniques do not provide rapid, high-throughput read-outs on protein localization, rendering them less than ideal in modern molecular science. For instance, the lack of antibody specificity in immunostaining, and potentially altering cell function during overexpression are some of the issues that need to be addressed. To this end, researchers have developed a new technique for single-cell labelling of endogenous proteins called CRISPR-Cas9-mediated homology-directed repair (SLENDR).
SLENDR is a powerful novel tool that allows for rapid and precise determination of protein subcellular localization. SLENDR was initially shown to correctly localize the position of CaMKIIα and CamKIIβ to the neuronal cytosol of embryonic mice. The researchers further demonstrated that this novel technique can accurately localize a wide spectrum of proteins, in a variety of cell types, with nanometer-scale resolution. For example, SLENDR enabled comparison of subcellular localization of proteins in different neuronal subtypes located in different brain regions across all developmental stages. SLENDR works by inducing double stranded breaks in the cell’s genome, which are then repaired via homology-directed repair using machinery that SLENDR introduces into the cell. The repaired DNA incorporates the genetic sequence for a tag that will be detected once the protein is translated.
Using this technique, researchers can label multiple different proteins with different tags in a single cell, thereby determining the subcellular location of each protein. Researchers further investigated how SLENDR can be used to elucidate protein-protein interactions. Here, knockout animals, animals lacking expression of a certain protein, are labelled for another endogenous protein using SLENDR. In doing so, researchers can determine the effects of the absent protein on the localization of the labelled protein. In conclusion, SLENDR is an incredible novel tool developed by researchers for determining subcellular localization. It provides accurate and rapid protein localization, high-throughput readouts, and is scalable to many endogenous proteins and cell types in the brain, at various different stages of brain development.
Written By: Haisam Shah, BSc