In the context of optimizing key genome editing procedures, researchers from the Department of Developmental Biology/Physiology at the Center for Organismal Studies at the University of Heidelberg have succeeded in improving the efficiency of molecular genetic methods such as CRISPR/Cas9 and related systems, and in expanding their fields of application. Together with colleagues from other disciplines, life scientists have refined these tools to enable, among other things, effective genetic screening for modeling specific genetic mutations. In addition, initially inaccessible DNA sequences can now be modified. According to Prof. Dr. Joachim Wittbrot, this opens up vast new areas of work in the field of basic research, and possibly therapeutic application.
Genome modification means the deliberate alteration of DNA using molecular genetic methods. It is used for plant and animal breeding, but also in basic medical and biological research. The most common ‘gene scissors’ include CRISPR/Cas9 and its variants known as base editors. In either case, the enzymes must be transported to the nucleus of the target cell. Upon arrival, the CRISPR/Cas9 system cuts DNA at specific locations, causing a double strand break. New pieces of DNA can then be inserted at this site. Base editors use a similar molecular mechanism but do not cut the double strand of DNA. Instead, the Cas9 protein-coupled enzyme performs a targeted exchange of nucleotides – the building blocks of the genome. In three consecutive studies, Prof. Wittbrot’s team has succeeded in significantly improving the efficiency and applicability of these methods.
The challenge when using CRISPR/Cas9 is the efficient delivery of the required Cas9 enzymes into the nucleus. “The cell has an elaborate ‘repelling’ mechanism. It distinguishes between proteins that are allowed to pass into the nucleus and those that are supposed to remain in the cytoplasm,” explains Dr. Tinatini Tavilides-Suk of Professor Wittbrot’s team. Access here is enabled by a tag of a few amino acids that acts like an “entrance ticket”. Scientists have now come up with a kind of generally valid “VIP ticket” that allows enzymes equipped with it to enter the nucleus very quickly. They called it the “High Efficiency Mark” and the “Hi Mark” for short. Other proteins that must penetrate the cell nucleus are also more successful with the hei-tag. In collaboration with pharmacologists from the University of Heidelberg, the team can show that Cas9 with respect to the ‘hei-tag’ could enable highly efficient and targeted genome modifications not only in the model organism medaka, Japanese rice fish (Oryzia latipis), but also in mammalian cell cultures and mouse embryos.
In another study, Heidelberg scientists showed that essential editors work very efficiently in the organism and are even suitable for genetic screening. In an experiment with Japanese ricefish, they were able to show that these locally limited targeted modifications to individual DNA building blocks achieve a result that can only be obtained through relatively laborious breeding of organisms with altered genes. The COS research team, in collaboration with Dr. Jakob Gerten, pediatric cardiologist at Heidelberg University Hospital, focused on some genetic mutations. These mutations are suspected to cause congenital heart defects in humans. By modifying individual DNA building blocks of relevant genes in the model organism, the scientists were able to simulate and study fish embryos with the described heart defects. The targeted intervention led to noticeable changes in the heart already during the early stages of embryonic development in the fish, say Bettina Wells and Dr Alex Cornian, two of the study’s first authors from Professor Wittbrot’s team. This enabled the researchers to confirm the original suspicion and establish a causal relationship between the genetic change and the clinical symptoms.
Precise interference with the genome of fish embryos is made possible by specially developed software ACEofBASEs, which are available online. It allows the identification of genomic loci that most efficiently lead to the desired changes in target genes and resulting proteins. Scientists say the Japanese rice fish is an excellent genetic model organism for modeling mutations like those identified from the patients involved. “Our method allows for efficient screening analysis and can therefore provide a starting point for the development of individualized medical treatment,” according to Jacob Gerten.
A third study, again from Wittbrodt’s group, addresses the limitations of primary editors. For such an editor to bind to the target cell’s DNA, there must be a certain sequence pattern. It’s called Protospacer Adjacent Motif, PAM for short. “If this conformation is not present near the building block of DNA to be altered, then it is impossible to exchange nucleotides,” explains Dr. Thumperger. A team under his direction has now found a way around this limitation. Two primary editors are used in one cell respectively. In an initial step, a new DNA-binding motif is generated for another primary editor, upon which this second editor, applied simultaneously, can edit a previously inaccessible site. This staggered use turned out to be highly efficient, explains Kaisa Pakari, first author of the study. With this trick, the Heidelberg scientists were able to increase the number of possible application sites for certified grammar editors by 65 percent. Now DNA sequences that were initially inaccessible can also be modified.
“The improvement of existing tools for genome editing and their precise application lead to very diverse possibilities for basic research and possibly new therapeutic approaches,” emphasizes Joachim Wittbrot.
The research results have been published in journals eLife And Development. The investigations were part of research carried out in the “3D Matter Made to Order” excellence group, which is jointly managed by the University of Heidelberg and the Karlsruhe Institute of Technology. The research studies and scientists involved were funded by the European Research Council, the German Research Foundation, the German Center for Cardiovascular Research, the German Heart Foundation and the Joachim Herz Foundation.