Genome editing of Schistosoma mansoni by integrating inducible CRISPR/Cas9 systems into genomic safe harbors and its application

• Applicant: Dr. Akito Koike, Ph.D.
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 534942530

The threats from infectious diseases generally have decreased, but some of them remain to be important. Especially, more effective control is required for several parasitic diseases. Parasites can be divided into protozoon, flatworms, and nematodes. Flatworms can be further divided into flukes and tapeworms. Schistosomiasis is one of such important parasitic diseases and its estimated mortality is over 14,000 annually. Schistosoma mansoni, one of the pathogens of schistosomiasis belongs to flukes. Techniques of genetic manipulation are basic requirements for reverse genetics. Since the appearance of CRISPR/Cas9, reverse genetics for non-model organisms have been developed greatly. However, such techniques for parasitic flatworms are rather limited. Brindley laboratory, the host laboratory of this proposal, has already succeeded in the genome editing of S. mansoni, but more precise regulation of genome editing will be required. Parasitic flatworms experience asexual reproduction in their intermediate hosts. These developmental stages in the intermediate hosts include phylotypic stages for parasitic flatworms. In the phylotypic stages of tapeworms and free-living flatworm planaria, anteroposterior axis decision factors play important roles in the development. We can conjecture that mechanisms should be similar also for flukes, but it has never been studied yet. In this project plan, I am going to develop inducible genome editing of S. mansoni by integrating drug-inducible CRISPR/Cas9 systems and sgRNA constructs into the genomic safe harbors. Genomic safe harbors are the regions in the genome far away from any known genes and so on. They can support the stable expression of the integrated transgenes without causing any detrimental effect on the original functions of the cells. One genomic safe harbor is already shown to be available by Brindley laboratory. but I will evaluate the availability of several more candidate regions. With the developed inducible genome editing systems, I will knock out the anteroposterior factors. In S. mansoni, it is only possible to integrate transgene constructs into the genome of the eggs. Because these genes are also necessary for embryogenesis, they should be knocked out in the latter stage. Therefore, an inducible knockout system is indispensable for this experiment of reverse genetics. In parallel, I will analyze the expression pattern of these anteroposterior factors in the phylotypic stages of S. mansoni and compare the results with those of planaria and tapeworms.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Paul J. Brindley, Ph.D.