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CRISPR to Reveal How “Water Bears” (Tardigrades) Survive Extreme Environments

Tardigrades are among the weirdest of animals.

Also known as “water bears” or “moss piglets,” the 1,300 recognized species are the only members of phylum Tardigrada, a term that means “slow stepper” for their somewhat waddling gait. German zoologist Johann August Ephraim Goeze first described the tardigrades in 1773. They live in seas, in fresh water, and on land.

Tardigrades are famous for hiding when environmental conditions turn treacherous, only to emerge years or even decades later unscathed. They survive extremes of dehydration, radiation, and great ranges of temperature and pressure. For example, tardigrades live under almost 6,000 times the pressure of the Earth’s atmosphere at sea level. They’re called “extremotolerant.”

They live pretty much everywhere, from mountain peaks to beneath glaciers and oceans and lakes, under the leaf carpet of forests, along logs and stones. To see them, collect a bit of lichen or moss and soak it overnight, then squeeze it onto a light microscope slide.

We could learn a lot about how tardigrades survive in the extremes. Now a report in PLoS Genetics from researchers at the University of Tokyo describes use of the gene-editing tool CRISPR to better understand the unusual traits of the just-barely-visible tardigrades.

Unusual Anatomy and Physiology Enables Survival

A tardigrade has 8 legs, a translucent covering, and is about the size of a period at the end of a sentence. The body is rotund and segmented, with a flattened, bulbous head.

They aren’t insects or arachnids, but share a common ancestor with these arthropods from about 500 million years ago. So tardigrades aren’t quite bugs, nor exactly worms, seeming to be largely head with a tiny tail. They’ve lost some segmentation genes over evolutionary time.

Under harsh environmental conditions, a tardigrade in survival mode rapidly dries out into a state of near-nothingness, its metabolism on hold, and waits out environmental conditions. This skill is called cryptobiosis, and the corpse-like form they assume is a “tun” state, their heads and legs retracted, the animal hiding. “Their metabolism slows to 0.01% of normal, and their water content drops to less than 1%,” according to 10 Astounding Facts About Tardigrades. Different tun states are adaptations to different environmental challenges.
As a tardigrade shrinks and goes into hiding, a sugar, trehalose, replaces the water in their cells with a gel-like consistency. A tun state can extend life span from under 3 years to up to 3 decades.

Tardigrades were obvious choices for animals to observe in space, surviving for awhile the vacuum, lack of gravity, extreme temperatures, and radiation of low-Earth orbit of NASA’S FOTON-M3 mission in 2007. Some managed to lay eggs, but others succumbed eventually to the prolonged, intense UV exposure and lack of oxygen.

The animals also tolerated conditions aboard the International Space Station in 2011, and seemed to have survived a crash into the moon from a probe in 2019. But like sea monkeys, the creatures would need liquid water to snap back to life. Imagine colonizing the moon only to discover the curious tardigrades already there. After all, they can reproduce even under radiation levels 1,000 times that which would kill a human, possessing admirable DNA repair skills.

An article in Scientific Reports from 2017 points out that tardigrades could be the sole surviving species of a global environmental disaster.

CRISPR Isolates Tardigrades’ Protective Genes

A classic approach of genetics is to identify a gene’s function by deleting or disarming it – historically using mutagens or radiation, or just awaiting mother nature. But since 2012, the gene editing tool CRISPR has introduced a specificity to removing a gene to see what it does. And that’s what the work described in the new paper in PLoS Genetics does.

The University of Tokyo team used CRISPR on “a highly resilient tardigrade species previously impossible to study with genome-editing tools,” according to a news release.

“To understand tardigrades’ superpowers, we first need to understand the way their genes function. My team and I have developed a method to edit genes — adding, removing or overwriting them — like you would do on computer data, in a very tolerant species of tardigrade, Ramazzottius varieornatus. This can now allow researchers to study tardigrade genetic traits as they might more established lab-based animals, such as fruit flies or nematodes,” said Takekazu Kunieda.

Tardigrades of the species are all female, and reproduce parthenogenetically (without a partner). Their genomes have two identical copies of each gene, which greatly eases editing.

The investigators used an approach developed for insects called “direct parental CRISPR,” or “DIPA-CRISPR,” which makes a genetic change that is passed to the animal’s offspring, no mating necessary. The tool can either “knock out” a gene to analyze which function is missing in the next generation, or “knock in” a gene to see what it does. And the experiments were straightforward: inject concentrated CRISPR solution into the animals shortly before they release their first eggs.

The fact that the species is parthenogenetic means that two copies of the same gene in an individual are always identical – and that means it isn’t necessary to set up and follow crosses to obtain a progeny class with a specific genotype. (As a former Drosophila geneticist, I can attest to the value.)

The ultimate goal is to proceed gene-by-gene to dissect the basis of the animal’s extraordinary protections. And specific discoveries may have practical applications. One gene, for example, enables the sugar trehalose to gum up the watery interiors of the tardigrade’s cells, facilitating survivable extreme dehydration. The researchers suggest that the chemistry of the trait might be applied to organ transplantation, making it possible to dehydrate and rehydrate a liver, heart, or kidney without inflicting damage. This could revolutionize the way organs are donated, transported, and used in surgery to save lives, the researchers write.

Another application of a tardigrade talent is the dry preservation of biomaterials, such as biologic drugs and vaccines.

Summed up Kunieda, “Tardigrades not only offer us a glimpse at what medical advances might be possible, but their range of remarkable traits means they had an incredible evolutionary story, one we hope to tell as we compare their genomes to closely related creatures using our new DIPA-CRISPR-based technique. CRISPR can be an incredible tool for understanding life and aiding in useful applications that can positively impact the world.”

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