Gene scissors in camouflage mode help in the search for cancer therapies

The immune system plays a crucial role in fighting tumours and metastases. Consequently, it is decisive to conduct cancer research in mouse models with an immune system that is as natural as possible – which is easier said than done.

Thanks to CRISPR/Cas9 technology, researchers can quickly create a pool of hundreds of tumour cells, each with a different gene switched off. When transplanted into mice, it becomes apparent which of these silenced genes influences the development and spread of cancer. Assisted by such CRISPR screens, scientists are able to identify valuable approaches for the development of new therapies.

But the method comes with a catch. The components of CRISPR/Cas9 originate mainly from bacteria, with the result that they are recognised as foreign by the immune system of mice and attacked. Researchers suspect that this reaction distorts the results of CRISPR screens.

The research group headed by Nicola Aceto, Professor of Molecular Oncology at ETH Zurich, has now demonstrated in detail for the first time that this is indeed the case. At the same time, the team has presented an elegant solution to the problem – a special strategy that lays a kind of molecular cloak over CRISPR/Cas9, making it invisible to the immune system. The results have just been published in the journal Cell.

Bacterial components interfere

Initially, the researchers used well-characterised mouse models for various types of cancer to investigate the effect of the bacterial components of CRISPR/Cas9 on the formation of tumours and metastases.

They found that tumour cells implanted in mice were rejected more often and were genetically less heterogeneous in the presence of CRISPR/Cas9 components. In addition, fewer metastases formed. Consequently, the immune system's response prevented the normal progression of cancer in the animal models. "We were surprised at how significantly this can distort the results of CRISPR screens," as Massimo Saini, first author of the study and ETH Pioneer Fellow in Aceto's group, stated. 

Invisibility cloak for screens

In response to this situation, the ETH team has now developed an alternative method for CRISPR/Cas9 screens that triggers virtually no immune response. In order to achieve this, the researchers exposed the tumour cells to the bacterial gene scissors Cas9 only temporarily. Moreover, they developed a method to isolate only those tumour cells in which a gene had been successfully silenced. These cells no longer contained Cas9 or any other elements that could trigger an immune response.

They also exchanged the so-called reporter genes. These are genes that are incorporated into the genetic material of the tumour cells in CRISPR screens instead of the silenced genes. Subsequently, the product of these genes enables the researchers to track modified tumour cells in the mice. Instead of the classic reporter genes, which originate from various organisms, a new gene is now used whose product differs only minimally from a protein that is produced naturally in the mice. This allows it to fly under the radar, so to speak, and remain undetected by the immune system. 

"We have developed a method for performing CRISPR screens in mice with intact immune systems, without incurring any undesirable side effects," summarises Prof. Aceto. The ingenious thing about this is the fact that the system is versatile and can also be used in humanised mice – animals that have a human immune system. "This is as close to cancer patients as you can get." Moreover, the cloak of invisibility for the gene scissors is also suitable for applications in personalised medicine or for research into autoimmune diseases. 

"With this system, we are now achieving a new level of accuracy and – particularly significant for us – we are able to discover new targets for therapies," says Saini. 

Overlooked genes for metastasis formation uncovered

The team has already performed a CRISPR screen with the cloaked version of the gene scissors and landed a highly promising hit: silencing two genes called AMH and AMHR2 drastically reduced the number of metastases in a mouse model for breast cancer.

Further investigations showed that the signalling pathway in which these two genes are involved is clinically relevant. For example, the evaluation of patient data revealed that high levels of AMH protein in the tumour are associated with more frequent relapses and higher mortality in breast cancer. Consequently, the AMH/AMHR2 gene pair represents a new approach to combating metastases.

"The significance of this signalling pathway has been underestimated," says Aceto. "Thanks to CRISPR in stealth mode, we are now able to uncover connections that were previously hidden."

References

Saini M, Castro-Giner F, Hotz A, et al. StealTHY: An immunogen-free CRISPR platform to expose concealed metastasis regulators in immunocompetent models, Cell (2025), https://doi.org/10.1016/j.cell.2025.10.007