What Is Gene Targeting and How Does It Work?

Gene targeting is a genetic technique that allows scientists to alter an organism’s DNA at a specific, predetermined location. By making controlled changes to the genetic code, researchers can investigate biological processes and the underlying causes of diseases. This technology is distinct from older methods that involved the random insertion of genetic material, as its precision allows for changes ranging from altering a single unit of DNA to inserting new genes.

This capability has made it a valuable technique in fields from basic biological research to the development of new medical therapies. The principles behind this method were recognized with the 2007 Nobel Prize in Physiology or Medicine.

Core Principles of Gene Alteration

Gene targeting operates by using a cell’s natural DNA repair mechanism, a process called homologous recombination. Scientists leverage this by introducing a custom-designed piece of DNA, known as a targeting construct, into a cell. This construct is engineered to contain the desired genetic change, flanked by sequences that are identical to the DNA on either side of the target gene.

These flanking regions, called homology arms, act as a guidance system, directing the construct to the precise location in the genome. The cell’s repair machinery recognizes the similarity between the construct and the native DNA sequence. It then initiates an exchange, swapping the original segment of the cell’s DNA with the engineered sequence from the targeting construct.

To increase the frequency of this exchange, scientists use molecular tools to create a targeted double-strand break in the cell’s DNA. This break acts as a signal, stimulating the cell’s repair systems and encouraging them to use the provided DNA construct as a template. This guided repair process ensures the genetic modification is made only at the intended site.

Common Gene Modification Outcomes

Gene targeting can achieve several specific outcomes, allowing researchers to study gene function in different ways. The primary applications include:

• Gene knockout, where a gene is completely inactivated or deleted. This allows researchers to observe the consequences of the gene’s absence, providing strong clues about its normal function.
• Gene knock-in, which introduces new genetic material at a specific locus. This can involve inserting a reporter gene, like Green Fluorescent Protein (GFP), to visually track a protein, or replacing a mouse gene with its human equivalent to create more relevant disease models.
• Point mutations, which change a single DNA letter. This is useful for modeling genetic diseases caused by subtle, specific mutations to study how a single change can alter a protein’s function.
• Conditional modifications, which use systems like Cre-Lox to alter a gene only in specific cell types or at certain times. This provides intricate control to study a gene’s function in a particular tissue or developmental stage.

Technologies in Gene Targeting

The ability to perform gene targeting has been advanced by technologies designed to cut DNA at specific locations. Early methods included Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). These are engineered proteins that bind to specific DNA sequences and cut them, initiating the homologous recombination process. However, designing these custom proteins for each new target was often complex.

The field was transformed by the development of the CRISPR-Cas9 system for its efficiency and ease of use. Adapted from a bacterial immune system, this technology uses two components: the Cas9 enzyme, which acts as “molecular scissors” to cut DNA, and a guide RNA (gRNA). The gRNA is a short, synthetic RNA molecule designed to be complementary to the target DNA sequence, guiding the Cas9 enzyme to the correct location.

The simplicity of designing a new gRNA for each target makes CRISPR-Cas9 more versatile than its predecessors. Upon reaching its target, the gRNA binds to the DNA, and the Cas9 enzyme creates a double-strand break. This break triggers the cell’s repair pathways, which can then be harnessed to achieve the desired genetic modification.

Impact on Scientific Discovery and Healthcare

Gene targeting has significantly impacted scientific research, primarily through the creation of animal models of human disease. By introducing disease-causing mutations into organisms like mice, researchers can study the progression of conditions such as cancer, cystic fibrosis, and Alzheimer’s in a living system. These models are used for understanding the molecular mechanisms of disease and for the initial testing of potential treatments.

The technology is also central to developing new medical treatments, particularly gene therapies. For genetic disorders caused by a single faulty gene, such as sickle cell anemia or Duchenne muscular dystrophy, gene targeting offers the potential to directly correct the mutation in a patient’s cells. Clinical trials are exploring this approach to provide one-time treatments for diseases that were once considered intractable.

In the pharmaceutical industry, gene targeting accelerates drug discovery. Genetically modified cell lines and animal models are used to screen new drug compounds and to understand how they work at a molecular level. For instance, a human gene can be knocked into a mouse to test a drug’s effectiveness and safety on the human version of a protein. This technology continues to drive innovation in biology, agriculture, and medicine.