An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

Genes are often referred to as the blueprints of life. Understanding the role of the genes in human cells is one of the major goals of biology. Recent advances in gene editing technologies, such as CRISPR/Cas9, mean scientists can now edit or delete precise sections within human genes, similar to how we edit words in a document on a computer. This has made it possible to insert small sequences that encode specific “tags” into genes. This in turn means that when a protein is built following the instructions in the gene, the protein includes the tag too, making it easy to monitor. Tags on proteins can help scientists understand what those proteins do by answering various questions, such as: where is the protein found in the cell? How much of the protein is there in each cell? Does this change as the cell matures? What does the protein interact with? Yet, more research could be done if the tagging process was made easier, quicker and more efficient. Dewari et al. have now come up with an improved gene editing approach that enabled them to rapidly tag hundreds of proteins all at the same time, with efficiencies that were much higher than expected based on previous approaches. The strategy uses common “off the shelf” reagents that can be designed with a new user-friendly, web-based tool called “Tag-IN”. Dewari et al. focused on optimizing their method in freshly grown stem cells, originally collected from mice and humans. They then went on to show the scalability and efficiency of this approach by tagging 60 different proteins in brain stem cells from mice. Now, rather than being limited to a handful of genes of interest, scientists can explore large families of genes in a variety of mouse and human cells in a much quicker and more comprehensive manner. Also, working with stem cells that can be freshly collected from individuals rather than cells that have been grown in the laboratory for a long time will be more useful for biological and disease studies. In the long-term, more knowledge of how protein-coding genes work in different human cells will benefit patients as new drugs or therapeutic targets are discovered.