In collaboration with the National Institute of Genetics, we sequenced the genome of Ramazzottius varieornatus (YOKOZUNA-1 strain), a tardigrade species known for its exceptionally high tolerance to rapid desiccation and extreme environments. The genome size is compact at 55 Mb—approximately 1/3 the size of a fruit fly's and less than 1/50 of the human genome. It contains an estimated 15,000 to 20,000 genes. While over half are shared with other animals, there is a notable expansion of stress-response genes involved in antioxidant defense and DNA repair. Horizontal gene transfer (HGT) accounted for less than 2% of the genome, which is not as high as previously speculated for tardigrades.
Approximately 40% of the total genes are taxonomically restricted to tardigrades. Protein-based analyses have revealed that many key tolerance proteins (described below) are encoded by these unique genes, suggesting they play a pivotal role in the organism's extreme resilience. (Hashimoto et al., Nat Commun, 2016)
High-dose radiation and complete desiccation cause severe DNA damage, including double-strand breaks (DSBs). To understand how R. varieornatus maintains DNA integrity, we identified a novel DNA-binding protein and named it Dsup (Damage suppressor). While Dsup is unique to tardigrades, its expression in cultured human cells reduced X-ray-induced DNA damage by approximately 40%. Furthermore, Dsup-expressing cells remained viable and capable of proliferation even after exposure to radiation doses that were lethal to ordinary cells. (Hashimoto et al., Nat Commun, 2016)
Subsequent studies by laboratories worldwide have confirmed that Dsup provides DNA protection in a wide variety of organisms, including Drosophila, C. elegans, yeast, rice, and tobacco.
Our lab has identified multiple families of tardigrade-specific protective proteins, including CAHS, SAHS, MAHS, and RvLEAM. Among these, CAHS (Cytoplasmic Abundant Heat-Soluble) proteins, first identified by our lab in 2012, have been most extensively characterized. These highly heat-resistant proteins are significantly upregulated during desiccation across many species. Multiple labs independently reported that CAHS proteins form filaments in vitro. We further demonstrated that CAHS proteins undergo fibrillization inside living cells in response to dehydration stress, leading to cellular stiffening and enhanced stress tolerance. (Tanaka A et al., PLOS Biol, 2022)
In 2024, we successfully established a long-awaited genome editing technique for tardigrades at the individual level. By injecting Cas9 RNP into the hemocoel of female adults (DIPA-CRISPR), we can efficiently obtain edited offspring. We have achieved not only gene knockouts but also knock-ins using ssODNs. Interestingly, the resulting individuals carry edited sequences homozygously. This is likely due to a unique meiotic mechanism specific to their parthenogenetic reproduction, making it a biologically fascinating phenomenon. (Kondo et al., PLOS Genet, 2024)
Since currently identified tolerance genes do not yet fully replicate the extreme resilience of tardigrades in other systems, we believe many undiscovered tolerance genes remain hidden in their genome. This technology is expected to dramatically accelerate our understanding of the molecular logic supporting tardigrade extremotolerance.
Tardigrade-specific tolerance genes discovered by our laboratory are available through the following biological resource centers (distribution is generally limited to research institutions):
・Addgene: Kunieda Lab
・RIKEN BRC: Takekazu Kunieda