Genetically modified animals play a major role in biomedical research. Mice are most commonly used because they are easy to breed and keep. Pigs are more similar to humans, but until now it has been time-consuming and inefficient to genetically modify, raise and reproduce such large animals. As a possible alternative, researchers have now created pigs and chickens that are already producing the Cas9 gene scissors in all the cells of their bodies. This makes it possible to edit the genes in living animals with little effort, instead of having to create a new animal model for each target gene.
The Crispr / Cas9 gene scissors allow targeted interventions on the DNA. By cutting and pasting, genes can be changed or inactivated with pinpoint accuracy. The system consists of two components: The so-called gRNA (guide RNA) is used to identify the correct DNA segment. This short sequence binds to the portion of a gene that is to be modified. The enzyme Cas9 nuclease, the actual gene scissors, binds to the gRNA and cuts the respective section of the target DNA. The method is one of the most important tools in genetic engineering and is easy and inexpensive to use. In order to produce genetically modified animals, e.g. for medical research, it is necessary to intervene in the germ line and to breed the animals produced in this way. This process is lengthy and inefficient, especially with larger animals.
Normally developed and fertile
Researchers led by Beate Rieblinger from the Technical University of Munich have now created pigs and chickens that already have a component of the Crispr / Cas9 system: “The animals we generate also provide the genetic scissors – the Cas9 protein,” explains Rieblinger’s colleague Benjamin Schusser. “All that remains is to introduce the conductive RNAs in order to obtain animals that have certain genetic properties.” In order to produce the Cas9-transgenic animals, the researchers introduced Cas9 into the genome of early pig and chicken embryos. The live-born animals in which the genetic modification had worked continued to reproduce them. This process took around three years.
“Both the Cas9-transgenic chickens and the pigs developed normally, showed no obvious abnormalities, for example in weight gain, and were fertile,” the researchers report. Because the Cas9 genes were introduced in the early stages of embryonic development, the germ cells and offspring of these animals also have the desired properties. “Cas9 can now be used in all developmental stages of the animals, since every cell in the body permanently possesses the Cas9 protein,” says Schusser. The researchers demonstrated this using samples from many different tissue types in the animals, including the heart, kidney, muscles, liver, lungs and brain.
Flexible application options
As a test, the researchers carried out various gene changes, both on chicken embryos and live pigs, and on cell samples that they had obtained from the Cas9 animals. The results confirmed: Just by adding the gRNA, the respective target genes could be processed in a relevant percentage of the cells. “The presence of Cas9 in the cells accelerates and simplifies the processes significantly,” says Riebinger’s colleague Angelika Schnieke. This enables an uncomplicated use in biomedical research. “Our group has an interest in modeling human cancers in pigs, such as colon cancer,” the researchers write. The newly created animal lines can help with this. “The animals equipped with Cas9 enable, for example, tumor-relevant genes to be specifically inactivated and the development of cancer to be simulated,” explains Schnieke.
Thanks to the flexible use of genetic manipulations, the researchers can also test directly on the animal which genes are involved in the development of disease resistance, for example. This is interesting for farm animal research, among other things. “Our Cas9-expressing chickens and pigs represent an innovative resource for genome editing in the biomedical and agricultural sciences,” summarizes Schnieke. They would also make the animals they bred available to other research groups. “Biomedical and agricultural research can thus be advanced through efficient genome editing in living animals.”
Source: Beate Rieblinger (Technical University of Munich) et al., Proceedings of the National Academy of Sciences, doi: 10.1073 / pnas.2022562118