Switching off or changing genes or inserting new genes at a specific location: Genome editing involves the targeted modification of DNA. It is used to breed plants and animals, but also in basic medical and biological research. The most common procedures include the “gene scissors” CRISPR/Cas9 and its variants known as base editors. In both cases, enzymes have to be transported into the nucleus of the target cell.
VIP admission ticket for the cell nucleus
In three successive studies, a team led by DZHK scientist Prof. Dr Joachim Wittbrodt from Heidelberg succeeded in considerably enhancing the efficiency and applicability of these methods.A challenge when using CRISPR/Cas9 consists in the efficient delivery of the required Cas9 enzymes to the nucleus. "The cell has an elaborate ‘bouncer’ mechanism. It distinguishes between proteins that are allowed to translocate into the nucleus and those that are supposed to stay in the cytoplasm,” explains Dr Tinatini Tavhelidse-Suck from Prof. Wittbrodt's team. Access is enabled here by a tag made up of a few amino acids that functions like an “admission ticket”.
The scientists have now come up with a kind of generally valid “VIP admission ticket” which lets enzymes equipped with it into the nucleus very quickly. They have named it “high efficiency-tag”, “hei-tag” for short. Cas9 in connection with the “hei-tag” ticket can enable highly efficient targeted genome alterations not only in the Japanese ricefish (Oryzias latipes), but also in mammalian cell cultures and mouse embryos.
Screening for congenital heart defects
In another study, the Heidelberg scientists have demonstrated that base editors are even suitable for genetic screening: In collaboration with DZHK scientist Dr Dr Jakob Gierten, a paediatric cardiologist at Heidelberg University Hospital, they looked at specific gene mutations suspected of triggering congenital heart defects in humans. In experiments with Japanese ricefish, they were able to establish a causal connection between genetic alteration and clinical symptoms.
Through modifying individual building blocks of the DNA of the relevant genes in the model organism, the scientists were able to imitate and study fish embryos with the described heart defects. The targeted intervention led to visible changes in the heart already during early stages of fish embryonic development. The Japanese ricefish is an excellent genetic model organism for modelling mutations like those identified from the respective patients. "Our method enables an efficient screening analysis and could therefore offer a starting point for developing individualised medical treatment," says Jakob Gierten.
Trick allows base editors to be used in previously inaccessible locations
In a third study, Prof. Wittbrodt's research group found a way to circumvent a restriction under which base editors do not work: For such an editor to bind to the DNA of the target cell at all, there has to be a specific sequence motif. With a trick, the scientists were able to increase the number of possible application sites of established base editors by 65 percent. Now DNA sequences that were initially inaccessible can also be modified. The trick is to use two base editors in a single cell one after the other. In an initial step, a new DNA binding motif for a further base editor is generated, upon which this second editor, which is applied simultaneously, can edit a site that was inaccessible before.
The research studies and scientists involved were funded by the European Research Council, the German Research Foundation, the German Centre for Cardiovascular Research, the German Heart Foundation, and the Joachim Herz Foundation.
Original publications:
T. Thumberger, T. Tavhelidse-Suck, J. A. Gutierrez-Triana, A. Cornean, R. Medert, B. Welz, M. Freichel, J. Wittbrodt: Boosting targeted genome editing using the hei-tag. eLife (25 March 2022), https://doi.org/10.7554/eLife.70558
A. Cornean, J. Gierten, B. Welz, J. L. Mateo, T. Thumberger, J. Wittbrodt: Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction. eLife (4 April 2022), https://doi.org/10.7554/eLife.72124
K. Pakari, J. Wittbrodt, T. Thumberger: De novo PAM generation to reach initially inaccessible target sites for base editing. Development (23 January 2023), https://doi.org/10.1242/dev.201115
