The tardigrade Dsup protein enhances radioresistance in Drosophila melanogaster and acts as an unspecific repressor of transcription

Zarubin, Mikhail; Azorskaya, Talyana; Kuldoshina, Olga; Alekseev, Sergey; Mitrofanov, Semen; Kravchenko, Elena

doi:10.1016/j.isci.2023.106998

Cited by 10 publications

(7 citation statements)

References 49 publications

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“…SSBs are non-specific in binding ssDNA 26 . Therefore, we envision a mechanism similar to what has been proposed for Dsup: protein can coat DNA to limit damage during genotoxic stresses, including desiccation 13,[38][39][40] . The potency of Dsup has been demonstrated in a number of contexts, yet it has also been reported to alter normal transcriptional regulation and may itself promote DNA damage in some contexts [40][41][42] .…”

Section: Discussionmentioning

confidence: 97%

A bacterial expression cloning screen reveals tardigrade single-stranded DNA-binding proteins as potent desicco-protectants

Hibshman,

Clark-Hachtel,

Bloom

et al. 2023

Preprint

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SummaryTardigrades are known for their ability to survive remarkable extremes, but relatively little is known about the molecular underpinnings that allow for their extraordinary stress tolerance. We devised an expression cloning approach to screen for novel protectants from desiccation-tolerant tardigrades. We expressed cDNA libraries from the tardigradesHypsibius exemplarisandRamazzottius varieornatusinE. coliand subjected the bacteria to desiccation. Sequencing the populations of surviving bacteria revealed enrichment of mitochondrial single-stranded DNA-binding proteins (mtSSBs) from both tardigrade species. Expression of mtSSBs in bacteria improved desiccation survival to similar levels as some of the best protectants known to date. The DNA-binding activity of the oligonucleotide/oligosaccharide binding (OB) fold domain of mtSSB was necessary and sufficient to improve desiccation tolerance. By comparing the efficacy of mtSSBs from multiple organisms, we provide evidence that desicco-protection is a conserved feature of mtSSBs. More broadly, any OB-fold containing protein may act similarly to promote desiccation tolerance, likely by coating ssDNA to limit damage during desiccation. These results identify single-stranded DNA-binding activity as a potent contributor to extreme desiccation tolerance.

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Section: Discussionmentioning

confidence: 97%

A bacterial expression cloning screen reveals tardigrade single-stranded DNA-binding proteins as potent desicco-protectants

Hibshman,

Clark-Hachtel,

Bloom

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Instead, recent studies suggested that tardigrades have and utilize their own unique protective proteins whose expression is abundant and/or significantly induced by desiccation during anhydrobiosis [13–16]. Their functions and roles in the resilient ability have been elucidated largely using heterologous expression and/or in vitro systems [14, 15, 17–22]. Although RNAi is feasible and has successfully been used for the elucidation of gene functions in some cases [17, 23, 24], the knock-down efficiency varied depending on the target genes and was not always sufficient.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Kondo,

Tanaka,

Kunieda

2024

Preprint

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Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, the genetic manipulation methods in 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 conductive to generating gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRIPSR (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. Through 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 establishment of a simple method for generating homozygous knock-out/knock-in individuals not only facilitates in vivo analyses of molecular mechanisms underpinning extreme tolerance but also opens avenues for exploring various topics, including Evo-Devo, in tardigrades.

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“…Instead, recently accumulating studies of tardigrades have suggested that they possess and utilize their own unique protective proteins whose expression is high and/or significantly induced upon desiccation during anhydrobiosis [ 14 – 17 ]. Owing to technological limitations, their functions and roles in tardigrade resilience have been elucidated largely using heterologous expression and/or in vitro systems [ 15 , 16 , 18 – 23 ]. Although RNAi is feasible for analyzing gene function and has been successfully used in some cases [ 18 , 24 , 25 ], the knockdown efficiency varied depending on the target gene and was not always sufficient.…”

Section: Introductionmentioning

confidence: 99%

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

Kondo,

Tanaka,

Kunieda

2024

PLoS Genet

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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.

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The tardigrade Dsup protein enhances radioresistance in Drosophila melanogaster and acts as an unspecific repressor of transcription

Cited by 10 publications

References 49 publications

A bacterial expression cloning screen reveals tardigrade single-stranded DNA-binding proteins as potent desicco-protectants

A bacterial expression cloning screen reveals tardigrade single-stranded DNA-binding proteins as potent desicco-protectants

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

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

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