Efficient way to study molecular mechanisms without creating new animal models

The world is continuously moving towards developing better healthcare for all living beings, which requires more efficient ways to study diseases and biomedical issues. This includes studies on genetically modified animals that researchers have been working on for years at the Technical University of Munich (TUM) lead by Benjamin Schusser, Angelika Schnieke, Christian Kupatt and Benedikt Kaufer (FU Berlin).

They created healthy and fertile transgenic animals – pigs and chickens, that constitutively express Cas9 in all organs. This allows genome editing in vivo in specific organs and tissues without the need to generate germline-modified animals, which is a quite laborious and time-consuming task.

CRISPR/ Cas9 is a gene editing technique that is used to change the genomes of living organisms. CRISPR is a family of repetitive DNA sequences originally found in bacteria, which are transcribed and processed to gRNAs in order to lead Cas9 to a specific DNA sequence. This system was adapted by researchers to directly deliver sequence-specific gRNAs. By delivering the Cas9 nuclease together with a guide RNA (gRNA) into a cell, the cell’s genome can be cut at a desired location, allowing existing genes to be removed and new ones to be added. There is also a possibility of simply turning genes on and off.

“This project was related to our previous work. We´re interested in generating genetically modified pigs and chickens for biomedical and agricultural research,” says professor Benjamin Schusser.

So far research has been mostly done on mouse models, since studying large animals has been proven to be quite difficult. Although other animals, such as pigs, resemble the human physiology more closely. In addition, studying species that are very different from humans, such as chickens, provides powerful insights into fundamental biological and biomedical processes.

Professor Schusser explains: “Until now, genome editing in a specific organ or tissue in a living animal has been a major challenge. Either complex genetic animal models had to be generated (conditional gene targeting), which is particularly costly and time-consuming in livestock. Or alternatively, the guide RNAs had to be introduced together with the Cas9 nuclease, where the size of the Cas9 nuclease and the limiting capacity of viral transfer vectors is a major obstacle, making the process very inefficient.”

They found an innovative and efficient way to eliminate this problem by generating animals that already express Cas9 and only require the introduction of guide RNAs, which are much smaller and therefore easier to introduce.

Cas9 transgenic pigs were generated by targeted placement of Streptococcus pyogenes Cas9 (SpCas9) at the ROSA26 locus, which has been shown to be a safe place for transgene expression without interrupting the function of essential genes. Cas9 transgenic chickens were generated by phiC31 integrase-mediated integration of an SpCas9 expression construct into a chicken pseudo attP site.

The transgene copy number was determined in both species by droplet digital PCR and revealed a single copy of SpCas9 in pigs and chickens. Both Cas9 transgenic chickens and pigs developed normally and were fertile.

Functionality of the SpCas9 transgene in vivo and in vitro was demonstrated using different methods for gRNA delivery like transfection with synthetic gRNAs, in ovo electroporation, or viral-based delivery methods. While all approaches resulted in editing of the target locus, the efficiency depended on the gRNA delivery method, ranging from 8% in vivo to 70% gene inactivation in vitro. These experiments showed that one can efficiently inactivate target genes or delete larger gene segments in a precise and organ-specific manner.

“In agriculture, genome-wide association studies (GWAS) can provide an indication of which genes or SNPs/polymorphisms may be involved in the formation of traits such as disease or heat resistance. Cas9 pigs and chickens now make it possible to test this directly in the animal. Furthermore, it is possible to use the mechanism of the CRISPR/Cas9 system to combat infections with DNA viruses. For example, initial work in cell culture has already shown elimination of Marek Disease Virus, a poultry herpes virus. In basic and biomedical research, we can introduce or correct mutations specifically in a desired organ without the need to generate new animal models for each target gene. This also reduces the number of experimental animals,” says professor Benjamin Schusser. He adds that these animals can be made available to other research groups, thus advancing biomedical and agricultural research internationally through efficient genome editing in live animals.

Transgenic pigs that express Cas9 in all of their organs. Picture taken by Andreas Heddergott/ TUM

Solis BioDyne product used: FIREPol® DNA Polymerase

Rieblinger, B., Sid, H., Duda, D., Bozoglu, T., Klinger, R., & Schlickenrieder, A. et al. (2021). Cas9-expressing chickens and pigs as resources for genome editing in livestock. Proceedings of the National Academy of Sciences, 118(10), e2022562118. doi: 10.1073/pnas.2022562118