By optimizing key genome editing procedures, researchers from the Department of Developmental Biology and Physiology at the University of Heidelberg Center for the Study of the Organism have succeeded in significantly increasing the effectiveness of molecular genetic methods such as CRISPR/Cas9 and related systems, and in expanding their fields of application. Together with colleagues from other disciplines, biological scientists have adapted these tools to include, among other things, efficient genetic screening to model specific gene mutations. In addition, initially inaccessible DNA sequences can now be modified. According to Prof. Dr. Joachim Wittbrodt, this opens up vast new areas of work in basic research and potentially in therapeutic applications.
Genome editing means the deliberate alteration of DNA by molecular genetic methods. It is used for plant and animal breeding, as well as in basic medical and biological research. The most common procedures involve CRISPR/Cas9 “gene scissors” and its variants known as base editors. In both cases, the enzymes must be transported into the nucleus of the target cell. Upon arrival, the CRISPR/Cas9 system cuts the DNA at specific locations, causing the two strands to break. New segments of DNA can then be inserted at this location. Base editors use a similar molecular mechanism, but they do not cut the DNA double strand. Instead, an enzyme connected to the Cas9 protein carries out a targeted exchange of nucleotides – the basic building blocks of the genome. In three consecutive studies, Prof. Wittbrodt’s team succeeded in significantly improving the efficiency and applicability of these methods.
A challenge in using CRISPR/Cas9 is to efficiently deliver the required Cas9 enzymes to the nucleus. “The cell has a sophisticated “ejector” mechanism. It distinguishes between proteins that are allowed to move into the nucleus and those that must remain in the cytoplasm,” explains Dr. Tinatini Tavhelidse-Sak from Prof. Witbrodt’s team. Access is enabled here by a tag that consists of a few amino acids and functions as an “entrance ticket”. Now scientists have come up with a kind of “VIP-ticket”, which very quickly lets the enzymes equipped with it into the nucleus. They called it “high efficiency tag” or “hey tag” for short. “Other proteins that have to enter the cell nucleus are also more successful with the hei-tag,” concludes Dr. Thomas Thumberger, who is also a researcher at the Center for Organism Research (COS). In collaboration with pharmacologists from Heidelberg University team was able to show that Cas9 in combination with a “hei-tag” ticket can allow highly efficient targeted genome changes not only in the model organism medaka, the Japanese rice fish (Oryzias latipes), but also in mammalian cell cultures and mouse embryos.
In a further study, the Heidelberg scientists showed that base editors work very efficiently in a living organism and are even suitable for genetic screening. In an experiment with Japanese rice fish, they were able to show that these locally limited targeted modifications in individual building blocks of DNA achieve a result that can otherwise only be obtained by relatively time-consuming breeding of organisms with altered genes. The COS research team, in collaboration with Dr. Jakob Geerten, a pediatric cardiologist at Heidelberg University Hospital, focused on certain genetic mutations. These mutations were thought to cause congenital heart defects in humans. By modifying individual DNA building blocks of the corresponding genes in a model organism, 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 at early stages of embryonic development in the fish, say Bettina Welz and Dr. Alex Cornean, two of the study’s first authors from Prof. Wittbrodt’s team. This allowed the researchers to confirm the initial suspicion and establish a causal relationship between genetic changes and clinical symptoms.
Precise intervention in the genome of fish embryos is made possible by specially designed ACEofBASEs software, which is available online. This allows the identification of genetic sites that are highly efficient in leading to desired changes in target genes and resulting proteins. Scientists say that the Japanese rice fish is an excellent genetic model of the organism for modeling mutations similar to those found in the corresponding patients. “Our method allows for an efficient screening assay and can therefore be a starting point for the development of individualized treatment,” said Jakob Geerten.
A third study, again from Wittbrodt’s group, deals with the limitations of base editors. For such an editor to bind the target cell’s DNA, a specific sequence motif must be present. It’s called Protospacer Adjacent Motif, PAM for short. “If this motif is missing near the DNA building block that needs to be changed, the exchange of nucleotides is not possible,” explains Dr. Tamberger. The team under his leadership found a way around this limitation. Two database editors in one cell are used consecutively. In the first step, a new DNA-binding motif is created for the downstream base editor, after which this second, concurrently applied editor can edit the previously inaccessible site. This phased use proved to be very effective, explains Kaisa Pakari, the first author of the study. With this trick, the Heidelberg scientists were able to increase the number of possible application sites generated by basic editors by 65 percent. Now, DNA sequences that were originally unavailable can also be modified.
“Optimization of existing genome editing tools and their precise application creates extremely diverse opportunities for basic research and potentially new therapeutic approaches,” emphasizes Joachim Wittbrodt.
The results of the study were published in journals eLife and Development. The research was part of research carried out at the 3D Matter Made to Order Cluster of Excellence, jointly managed by the University of Heidelberg and the Karlsruhe Institute of Technology. The research and participation of the scientists was funded by the European Research Council, the German Research Foundation, the German Center for Cardiovascular Research, the German Heart Foundation and the Joachim Hertz Foundation.