The effectiveness of molecular genetic techniques like CRISPR/Cas9 and related systems has been significantly improved, and their range of applications has been expanded, by researchers from the Department of Developmental Biology/Physiology at the Centre for Organismal Studies of Heidelberg University.
The life scientists improved these techniques in collaboration with colleagues from other fields to facilitate, among other things, efficient genetic screening for modeling certain gene mutations. Additionally, it is now possible to alter previously inaccessible DNA sequences. Prof. Dr Joachim Wittbrodt claims that this opens up a wide range of new topics for studies in basic research and, possibly, therapeutic application.
Genome editing is the deliberate manipulation of DNA by molecular genetic methods. In addition to being employed in biological and basic medical research, it is also used to breed plants and animals.
The “gene scissors” CRISPR/Cas9 and its variants, known as base editors, are among the most widely used techniques. Enzymes must be delivered to the target cell’s nucleus in both situations. The CRISPR/Cas9 system first arrives and uses precise locations to cut the DNA, leading to a double strand break.
New DNA segments can then be introduced at that site. Base editors do not cut the DNA double strand but rather employ a comparable molecular mechanism. Instead, a targeted exchange of nucleotides—the fundamental units of the genome—is carried out by an enzyme working in tandem with the Cas9 protein.
Prof. Wittbrodt’s team was successful in substantially increasing the effectiveness and application of these methodologies over the course of three subsequent research.
Delivery of the necessary Cas9 enzymes to the nucleus effectively presents a hurdle when employing CRISPR/Cas9.
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.”
Dr Tinatini Tavhelidse-Suck, Heidelberg University
Dr Tinatini Tavhelidse-Suck is a member of Prof. Wittbrodt’s team.
A tag comprised of a few amino acids that serve as an “admission ticket” allows access here. The researchers have now developed a sort of universally accepted “VIP admission ticket” that allows enzymes fitted with it to enter the nucleus very swiftly. It was named “high efficiency-tag,” or shortly “hei-tag.”
Other proteins that have to penetrate the cell nucleus are also more successful with ‘hei-tag.’”
Dr Thomas Thumberger, Researcher, Centre for Organismal Studies, Heidelberg University
In collaboration with pharmacologists from Heidelberg University, the team was able to demonstrate that Cas9 in combination with the “hei-tag” ticket can facilitate highly effective, targeted genome alterations in mammalian cell cultures and mouse embryos in addition to the model organism medaka, the Japanese ricefish (Oryzias latipes).
The Heidelberg researchers demonstrated that base editors function efficiently in living organisms and are even appropriate for genetic screening in a subsequent investigation. They were able to demonstrate in an experiment using Japanese ricefish that these localized, targeted changes in certain DNA building blocks produce a result that would normally require the comparatively time-consuming breeding of organisms with altered genes.
The COS research group concentrated on certain genetic mutations in collaboration with Dr Jakob Gierten, a pediatric cardiologist at Heidelberg University Hospital. These mutations were thought to be the cause of human congenital cardiac abnormalities. Researchers were able to mimic and examine fish embryos with the identified heart abnormalities by altering certain DNA strands in the model organism that contained the necessary genes.
According to Bettina Welz and Dr Alex Cornean, two of the first authors of the study from Prof. Wittbrodt’s team, the focused intervention resulted in noticeable changes in the heart already during the early stages of fish embryonic development. This allowed the researchers to establish a causal link between a genetic mutation and clinical symptoms and corroborate their first suspicion.
Through the use of specifically created software called ACEofBASEs, which is available online, the exact intervention in the fish embryos’ genomes was made possible. It makes it possible to pinpoint the genomic regions that modify the target genes and the resulting proteins in the desired ways extremely effectively.
According to the researchers, the Japanese ricefish is a great genetic model organism for simulating mutations similar to those seen in the respective patients.
“Our method enables an efficient screening analysis and could therefore offer a starting point for developing individualized medical treatment,” states Jakob Gierten.
The limitations of base editors are the subject of a third study, also from the Wittbrodt group. Such an editor requires a specific sequence motif in order to bind the DNA of a target cell. Protospacer Adjacent Motif, or PAM, is the short name for it.
If this motif is lacking near the DNA building block to be changed, it is impossible to exchange nucleotides.”
Dr Thomas Thumberger, Researcher, Centre for Organismal Studies
Now, a group under his leadership has discovered a solution for this constraint. In a single cell, two base editors are employed successively. In an initial phase, a new DNA binding motif for a subsequent base editor is formed, upon which this second editor, which is deployed concurrently, can edit a site that was inaccessible before.
Kaisa Pakari, the study’s first author, adds that this staggered use proved to be very effective. The Heidelberg scientists increased the number of potential application sites for existing base editors by 65% using this method. Now it is possible to alter DNA sequences that were previously unavailable.
“Optimizing the existing tools for genome editing and their fine-tuned application results in enormously varied possibilities for basic research and, potentially, novel therapeutic approaches,” concludes Joachim Wittbrodt.
- Thumberger, T., et al. (2022) Boosting targeted genome editing using the hei-tag. eLife. doi.org/10.7554/eLife.70558.
- Cornean, A., et al. (2022 Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction. eLife. doi.org/10.7554/eLife.72124.
- Pakari, K., et al. (2023) De novo PAM generation to reach initially inaccessible target sites for base editing. Development. doi.org/10.1242/dev.201115.