Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR.

Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, methods for genetically manipulating tardigrades have long been desired. Despite our prior success in somatic cell gene editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions that led to the generation of gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRISPR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. This establishment of a straightforward method for generating homozygous knockout/knock-in individuals not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades.

Application of CRISPR/Cas9 system and the preferred no-indel end-joining repair in tardigrades.

Tardigrades are small aquatic animals known for the tolerant ability against various extreme stresses. Recent studies identified several tardigrade-unique proteins as protective factors of biomolecules from extreme stresses. Due to the limitation of the technique available in tardigrades, the function of these protective molecules has largely been studied utilizing the systems of in vitro and the heterologous expression in other organisms. Although RNAi is feasible in tardigrades, their effects are variable and not always sufficient. To analyze the functions of the tardigrade protective proteins, in vivo genetic manipulations have been desired. In this study, we used a tardigrade Hypsibius exemplaris as a model whose genome is available, and developed the delivery method of Cas9 ribonucleoproteins (RNPs) to adult tardigrade cells. Cas9 RNPs containing two kinds of crRNAs were injected to the body cavity of adult tardigrades and subjected to the subsequent electroporation to facilitate the incorporation of RNPs to the cells. Using this delivery method, we detected the deletion of the intervening region between two crRNAs from the genome. Intriguingly, all examined joining sites exhibited no incorporation of insertions/deletions (indels), suggesting that no-indel end-joining is dominant repair system in this tardigrade. We also detected similar removal of the intervening region even in the tardigrades injected with Cas9 RNPs without electroporation and in this case the no-indel end-joining is detected in still dominant but not all examined joining sites. This study provides the development of the delivery method of Cas9 RNPs to tardigrade cells and our data also suggested that simultaneous application of more than two crRNAs/gRNAs are recommended to disrupt the target gene by CRISPR/Cas9 system to avoid scarless repair in the tardigrade.

Parallel evolution of trehalose production machinery in anhydrobiotic animals via recurrent gene loss and horizontal transfer.

Trehalose is a versatile non-reducing sugar. In some animal groups possessing its intrinsic production machinery, it is used as a potent protectant against environmental stresses, as well as blood sugar. However, the trehalose biosynthesis genes remain unidentified in the large majority of metazoan phyla, including vertebrates. To uncover the evolutionary history of trehalose production machinery in metazoans, we scrutinized the available genome resources and identified bifunctional trehalose-6-phosphate synthase-trehalose-6-phosphate phosphatase (TPS-TPP) genes in various taxa. The scan included our newly sequenced genome assembly of a desiccation-tolerant tardigrade Paramacrobiotus sp. TYO, revealing that this species retains TPS-TPP genes activated upon desiccation. Phylogenetic analyses identified a monophyletic group of the many of the metazoan TPS-TPP genes, namely 'pan-metazoan' genes, that were acquired in the early ancestors of metazoans. Furthermore, coordination of our results with the previous horizontal gene transfer studies illuminated that the two tardigrade lineages, nematodes and bdelloid rotifers, all of which include desiccation-tolerant species, independently acquired the TPS-TPP homologues via horizontal transfer accompanied with loss of the 'pan-metazoan' genes. Our results indicate that the parallel evolution of trehalose synthesis via recurrent loss and horizontal transfer of the biosynthesis genes resulted in the acquisition and/or augmentation of anhydrobiotic lives in animals.

Pre-treatment with D942, a furancarboxylic acid derivative, increases desiccation tolerance in an anhydrobiotic tardigrade Hypsibius exemplaris.

The tardigrade Hypsibius exemplaris can undergo anhydrobiosis. Several chemicals that inhibit successful anhydrobiosis in H. exemplaris have been identified, and these chemicals inhibit the activity of signaling molecules. In the present study, we investigated whether upregulation of the activity of these signaling molecules could improve desiccation tolerance of H. exemplaris. Pre-treatment with an indirect activator of AMP-activated protein kinase [AMPK; which directly inhibits mammalian NAD(P)H dehydrogenase [quinone] 1 [NQO1] of mitochondrial complex I (D942)] significantly improved desiccation tolerance of H. exemplaris, whereas a direct activator of AMPK did not. To elucidate the underlying molecular mechanisms, we examined the proteome of tardigrades treated with D942. Two proteins, putative glutathione S-transferase and pirin-like protein, were upregulated by treatment. Both of these proteins are known to be associated with the response to oxidative stress. One of the downregulated proteins was serine/threonine-proteinphosphatase 2A (PP2A) 65-kDa regulatory subunit A alpha isoform, and it is interesting to note that PP2A activity was previously suggested to be required for successful anhydrobiosis in H. exemplaris. Taken together, our results suggest that D942 treatment may partially induce responses common to those of desiccation stress. The identification of a chemical that improves desiccation tolerance of H. exemplaris may facilitate further investigation into desiccation tolerance mechanisms.

AMPK activity is required for the induction of anhydrobiosis in a tardigrade Hypsibius exemplaris, and its potential up-regulator is PP2A.

The anhydrobiotic tardigrade, Hypsibius exemplaris, was previously considered to require de novo gene expression and protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activity for successful anhydrobiosis. These indicate that H. exemplaris has signal transduction systems responding to desiccation stress, with the involvement of phosphorylation events. To this end, we carried out time-series phosphoproteomics of H. exemplaris exposed to mild desiccation stress and detected 48 phosphoproteins with significant differential regulations. Among them, immediate and successive reduction of phosphorylation levels of AMP-activated protein kinase (AMPK) was observed. The subsequent chemical genetic approach showed that AMPK was activated during the preconditioning stage for anhydrobiosis, and inhibition of its activity impaired successful anhydrobiosis. As PP2A is known to dephosphorylate AMPK in other organisms, we suggested that decreased phosphorylation levels of AMPK upon mild desiccation stress were caused by dephosphorylation by PP2A. Accordingly, phosphoproteomics of animals pre-treated with the PP1/PP2A inhibitor cantharidic acid (CA) lacked the decrease in phosphorylation levels of AMPK. These observations suggest that AMPK activity is required for successful anhydrobiosis in H. exemplaris, and its phosphorylation state is possibly regulated by PP2A.

Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.

Suggested Involvement of PP1/PP2A Activity and De Novo Gene Expression in Anhydrobiotic Survival in a Tardigrade, Hypsibius dujardini, by Chemical Genetic Approach.

Upon desiccation, some tardigrades enter an ametabolic dehydrated state called anhydrobiosis and can survive a desiccated environment in this state. For successful transition to anhydrobiosis, some anhydrobiotic tardigrades require pre-incubation under high humidity conditions, a process called preconditioning, prior to exposure to severe desiccation. Although tardigrades are thought to prepare for transition to anhydrobiosis during preconditioning, the molecular mechanisms governing such processes remain unknown. In this study, we used chemical genetic approaches to elucidate the regulatory mechanisms of anhydrobiosis in the anhydrobiotic tardigrade, Hypsibius dujardini. We first demonstrated that inhibition of transcription or translation drastically impaired anhydrobiotic survival, suggesting that de novo gene expression is required for successful transition to anhydrobiosis in this tardigrade. We then screened 81 chemicals and identified 5 chemicals that significantly impaired anhydrobiotic survival after severe desiccation, in contrast to little or no effect on survival after high humidity exposure only. In particular, cantharidic acid, a selective inhibitor of protein phosphatase (PP) 1 and PP2A, exhibited the most profound inhibitory effects. Another PP1/PP2A inhibitor, okadaic acid, also significantly and specifically impaired anhydrobiotic survival, suggesting that PP1/PP2A activity plays an important role for anhydrobiosis in this species. This is, to our knowledge, the first report of the required activities of signaling molecules for desiccation tolerance in tardigrades. The identified inhibitory chemicals could provide novel clues to elucidate the regulatory mechanisms underlying anhydrobiosis in tardigrades.