 Every biological process can be traced back to genes, and in many cases specific diseases can be linked to mistakes in genes. Understanding the exact role a gene plays in biology or disease is challenging, however, because multicellular organisms like humans are complex. Just over 30 years ago, a group of scientists made a pivotal discovery that led to the development of an important tool that changed the way scientists could study the role of a single gene and its impact on an entire biological system, the knockout mouse. You can think of a knockout mouse like baking a cake with an ingredient removed. Removing even one ingredient can lead to a cake that doesn't rise properly or isn't sweet enough. Similarly, taking away a single gene in an organism can reveal what key ingredient that gene provides to making a fully functional organism. While the theory is simple, the process of making a knockout mouse is complex and took decades to develop. To make a knockout mouse, researchers first isolate embryonic stem cells, ES cells, from an early mouse embryo, one of the earliest developmental stages of any organism. Most ES cells are a type of cell that still have the potential to develop into any cell or tissue in the body. So if a gene is removed from them, then all subsequent cells and tissues will also be devoid of the gene. One of the most common ways scientists can remove a gene from ES cells is using a method called homologous recombination. In this process, a piece of DNA containing identical genetic sequences to the region surrounding the gene of interest is used to swap out genetic material in the ES cell by either rendering the gene dysfunctional with the addition of a mutation or replacing it with some DNA that interferes with the gene. After the gene is removed, the ES cells are inserted into a mouse embryo and implanted into the uterus of a female mouse. The resulting baby mice will have some tissues where the desired gene is removed, those originating from the ES cell, and others where it is not, those originating from the embryo itself. These mice are known as chimeric mice, named after an ancient Greek beast, the chymera, consisting of body parts from several different animals. To generate mice where the target gene is gone in all cells, the partial knockout mouse is bred with a normal mouse. The resulting mouse litter will have some mice where one copy of the target gene is removed, also called heterozygotes. These heterozygotes are further bred together over several generations to produce a line of mice where the desired gene is completely removed, also known as homozygous knockouts. This discovery of how to make a knockout mouse was so critical that the three scientists involved won the Nobel Prize in Physiology or Medicine in 1997. Since then, knockout mice have been critical contributors to numerous scientific advances. Given humans and mice are identical in 85% of genes that make proteins, studying a gene's role in a mouse can provide critical insights to human biology and disease. One of the first examples was the creation of a mouse model for cystic fibrosis, a disease caused by a mutation in the CFTR gene. Knocking out this one gene in mice, recapitulated many of the cystic fibrosis symptoms observed in humans, allowing scientists to understand the complexities of this disease, ultimately helping us treat the same condition in humans. Since then, mouse models have advanced research for numerous diseases, including arthritis, various cancers, and Parkinson's disease. In recent years, knockout mouse technology has evolved considerably, most recently with the advent of CRISPR-Cas9. CRISPR-Cas9 is based on a naturally occurring immune system and bacteria to target and change specific genetic locations. This method provides several benefits. One, it can edit genes within an embryo itself, which can bypass the need for ES cells. This also allows editing in animal models other than mice, as ES cells are not available for most animals. Two, it is extremely precise and can edit multiple genetic locations simultaneously. And three, it can create a knockout mouse in about six months, compared to the nearly two years more traditional methods require. As with any scientific method, knockout mice also have certain drawbacks. Some genes, for example, are so essential that removing them leads to the death of the embryo or a mouse that cannot fully develop. Additionally, many biological processes or diseases are impacted or driven by more than one gene. So removing a single one might not replicate all disease symptoms. Lastly, there are important biological and genetic differences between mice and humans. So observations made in mice may not always translate to humans. Regardless, from the time they were first introduced, knockout mice have become an indispensable part of many biological laboratories around the world, contributing to nearly every scientific medical advance. Now, knockout technology is used in many different types of model organisms, for instance flies, worms and fish, further revolutionizing scientific research.