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CRISPR: the next generation of genome editing tools

CRISPR: the next generation of genome editing tools | SciFrye | Scoop.it

An arms race has been waged between bacteria and bacteriophage that would bring a satisfactory tear to Sun Tzu’s eye. Scientists have recently recognized that countermeasures developed by bacteria (and archaea) in response to phage infections can be retooled for use within molecular biology. In 2013, large strides have been made to co-opt this system (specifically and most commonly from Streptococcus pyogenes) for use in mammalian cells. This countermeasure, CRISPR (clustered regularly interspaced short palindromic repeats), has brought about another successive wave of genome engineering initiated by recombineering and followed more recently by zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).

 

ZFNs and TALENs perform a similar function yet the learning curve appears to be more difficult for development due to the use of protein-DNA contacts rather than the simplicity of designing RNA-DNA homology contacts. Although the potential for CRISPR in regards to genome editing within mammalian cells will be of greatest interest to the reader, the CRISPR backstory is equally compelling. Just as we have evolved immune responses to pathogens, so too have bacteria. CRISPR is an adapted immune response evolved by bacteria to create an immunological memory to ward off future phage infections. When a phage infects and injects its DNA within a bacterium, the DNA commandeers bacterial proteins and enzymes for use towards lytic or lysogenic phases. However, exposure of phage DNA allows the bacterium to copy and insert snippets (called spacers) of phage DNA into its genomic DNA between direct repeats (DR). These snippets can later be expressed as an operon (pre-CRISPR RNA, pre-crRNA) alongside a trans-activating CRISPR RNA (tracrRNA) and an effector CRISPR associated nuclease (Cas). Together these components surveil for foreign crRNA cognate sequence and cleave the targeted sequence.

 

Although hallmarks of CRISPR have been known since the late 80’s (CRISPR timeline) and was acronymed in 2002, Jinek et al. in August 2012 were the first to suggest the suitability of CRISPR towards genome editing. In February of 2013, Feng Zhang’s and George Church’s labs simultaneously published the first papers describing the use long oligos/constructs for editing via CRISPR in mammalian cells and made their plasmids readily available on Addgene. Zhang’s lab went one step further and has supplemented their papers with a helpful website and user forum. They have even gone so far as to publish a methods paper to streamline the use of their plasmids towards a plug-and-play, modular cloning approach with your target sequence of interest.

 

CRISPR works fairly well out of the box yet still has some imperfections that are being addressed. For example, CRISPR relies upon a protospacer adjacent motif (PAM; S. pyogenes sequence: NGG) 3’ to the targeting sequence to permit digestion. Although the ubiquity of NGG within the genome may seem advantageous, it may be limiting in some regions. Other species make use of different PAM sites that can be considered when choosing a cut sites of interest. Since double-stranded cuts could potentially create DNA lesions (a byproduct of the cell using non-homologous end joining [NHEJ] instead of homologous recombination) some labs are choosing to use modified Cas enzymes that nick DNA, instead of creating a double-strand break. This potential weakness of CRISPR to create DNA lesions via NHEJ, however, has been exploited by Eric Lander’s and Zhang’s lab this month (Jan. 2014). They have capitalized on the cell’s use of NHEJ to manufacture DNA lesions (frameshift mutations) at cut sites within genes on a large scale as a means to perform large genetic screens. Using this technique knocks out a gene and has the obvious advantage of fully ablating a gene’s expression compared to RNAi where some residual expression can be expected.

 

The advantages of CRISPR lends itself to future therapies. High efficiency, low-to-no background mutagenesis and easy construction put CRISPR front and center as the tool de jour for gene therapy. In combination with induced pluripotent stem cells (iPSCs), one can imagine the creation of patient-specific iPSCs created with non-integrative iPSC vectors and modified by CRISPR, devoid of any residual DNA footprint left behind by the iPSC vector or CRISPR correction. In conjunction with whole genome sequencing, genetically clean cell lines can be selected that are suitable for differentiation towards the germ layer of interest for subsequent autologous transplantation. Proof of principle experiments have already been published in models of cystic fibrosis and cataracts.

 

For better or worse, CRISPR is catching on like wildfire with young investigators, as noted recently by Michael Eisen. What may be looming in the future and not as openly discussed at this time is the potential for CRISPR to open up the genome to large scale editing. We tend to think of any particular genome as fairly static with slight variations between any two individuals and increased variation down the evolutionary line. However, CRISPR has proven to be a fantastic multitasker, capable of modifying multiple loci in one fell swoop as demonstrated by the Jaenisch lab (five loci). With the creation of Caribou Biosciences and a surprising round of venture capital raised by a powerhouse team at Editas Medicine in November ($43 million), CRISPR appears to also have sparked an interest in the private sector. With large sums of money at their disposal, these companies can now begin to look at the genome, not as a static entity, but more akin to operating system, a code that now has a facile editing tool. George Church, an Editas co-founder, has speculated in the past about the potential use of the human genome as the backbone for recreating the Neanderthal genome in his recent book and interview with "Der Spiegel". In an era where the J. Craig Venter Institute can create an organism’s genome de novo and a collaboration between Synthetic Genomics and Integrated DNA Technologies has proposed to synthesize DNA upwards of 2Mbp, the combination of CRISPR, synthetic DNA and some elbow grease will make the genome more accessible and Church’s speculations a potential reality.


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The man who planted a tree and grew a whole family of forests

The man who planted a tree and grew a whole family of forests | SciFrye | Scoop.it
As Brazil’s deforestation begins to climb again, one man has spent over 40 years planting a forest of his own

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A beachrock

A beachrock | SciFrye | Scoop.it
Researchers in the UPV/EHU's Department of Analytical Chemistry have published a study in which they analyze beachrocks, cemented sand formations that have industrial waste, produced as a result of metallurgical activities, trapped inside them. These strange rocks bear witness to the impact of industrial development and its influence on the coastal environment.

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VPNs Won’t Save You from Congress’ Internet Privacy Giveaway

VPNs Won’t Save You from Congress’ Internet Privacy Giveaway | SciFrye | Scoop.it
Get ready to say good-bye to your online privacy. Not that you ever really had it anyway.
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Researchers create artificial materials atom-by-atom

Researchers create artificial materials atom-by-atom | SciFrye | Scoop.it
Researchers at Aalto University have manufactured artificial materials with engineered electronic properties. By moving individual atoms under their microscope, the scientists were able to create atomic lattices with a predetermined electrical response. The possibility to precisely arrange the atoms on a sample bring 'designer quantum materials' one step closer to reality. By arranging atoms in a lattice, it becomes possible to engineer the electronic properties of the material through the atomic structure.

Working at a temperature of four degrees Kelvin, the researchers used a scanning tunnelling microscope (STM) to arrange vacancies in a single layer of chlorine atoms supported on a copper crystal.

"The correspondence between atomic structure and electronic properties is of course what happens in real materials as well, but here we have complete control over the structure. In principle, we could target any electronic property and implement it experimentally", says Dr. Robert Drost who carried out the experiments at Aalto University.

Using their atomic assembly method, the research team demonstrated complete control by creating two real-life structures inspired by fundamental model systems with exotic electronic properties.

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World’s First 'Menstrual Cycle in a Dish' Simulates Female Body

World’s First 'Menstrual Cycle in a Dish' Simulates Female Body | SciFrye | Scoop.it
After decades of drug testing on mostly men, a new device may help tailor treatments for different genders.
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Ubiquitous marine organism co-evolved with other microbes, promoting more complex ecosystems

Ubiquitous marine organism co-evolved with other microbes, promoting more complex ecosystems | SciFrye | Scoop.it
William Blake may have seen a world in a grain of sand, but for scientists at MIT the smallest of all photosynthetic bacteria holds clues to the evolution of entire ecosystems, and perhaps even the whole biosphere.

The key is a tiny bacterium called Prochlorococcus, which is the most abundant photosynthetic life form in the oceans. New research shows that this diminutive creature's metabolism has evolved in a way that may have helped trigger the rise of other organisms, to form a more complex marine ecosystem. Its evolution may even have helped to drive global changes that made possible the development of Earth's more complex organisms.

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Spinach Leaf Transformed Into Beating Human Heart Tissue

Spinach Leaf Transformed Into Beating Human Heart Tissue | SciFrye | Scoop.it
Using the plant like scaffolding, scientists built a mini version of a working heart, which may one day aid in tissue regeneration.
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Periodic Table of Mathematical Objects: The L-functions and Modular Forms Database

Periodic Table of Mathematical Objects: The L-functions and Modular Forms Database | SciFrye | Scoop.it

A team of more than 80 mathematicians from 12 countries has begun charting the terrain of rich, new mathematical worlds, and sharing their discoveries on the Web. The mathematical universe is filled with both familiar and exotic items, many of which are being made available for the first time.

 

The "L-functions and Modular Forms Database," abbreviated LMFDB, is an intricate catalog of mathematical objects and the connections between them. Making those relationships visible has been made possible largely by the coordinated efforts of a group of researchers developing new algorithms and performing calculations on an extensive network of computers. The project provides a new tool for several branches of mathematics, physics, and computer science.

 

A "periodic table" of mathematical objects

Project member John Voight, from Dartmouth College, observed that "our project is akin to the first periodic table of the elements. We have found enough of the building blocks that we can see the overall structure and begin to glimpse the underlying relationships." Similar to the elements in the periodic table, the fundamental objects in mathematics fall into categories. Those categories have names like L-function, elliptic curve, and modular form. The L-functions play a special role, acting like 'DNA' which characterizes the other objects. More than 20 million objects have been catalogued, each with its L-function that serves as a link between related items. Just as the value of genome sequencing is greatly increased when many members of a population have been sequenced, the comprehensive material in the LMFDB will be an indispensible tool for new discoveries.

 

The LMFDB provides a sophisticated web interface that allows both experts and amateurs to easily navigate its contents. Each object has a "home page" and links to related objects, or "friends." Holly Swisher, a project member from Oregon State University, commented that the friends links are one of the most valuable aspects of the project: "The LMFDB is really the only place where these interconnections are given in such clear, explicit, and navigable terms. Before our project it was difficult to find more than a handful of examples, and now we have millions."


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Researchers just uncovered a simple way to help combat the opioid epidemic

Researchers just uncovered a simple way to help combat the opioid epidemic | SciFrye | Scoop.it
It’s not the only answer, but it could be part of the solution to the deadliest drug crisis in US history.
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Face of Cambridge man brought to life 700 years after his death

Face of Cambridge man brought to life 700 years after his death | SciFrye | Scoop.it
Reconstruction is part of research project aimed at gaining insights into the anonymous poor of the medieval city
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Deadly spider venom could ward off stroke brain damage, say doctors

Deadly spider venom could ward off stroke brain damage, say doctors | SciFrye | Scoop.it


Ingredient in funnel web spider venom can protect cells from being destroyed by a stroke, even when given hours after the event, study shows

The protective molecule was discovered by chance as researchers sequenced the DNA of toxins in the venom of Hadronyche infensa, the Darling Downs funnel web spider. Photograph: Mark Baker/Reuters

Ian Sample Science editor
@iansample
Monday 20 March 2017 15.00 EDT Last modified on Wednesday 22 March 2017 13.32 EDT


Doctors have stumbled on an unlikely source for a drug to ward off brain damage caused by strokes: the venom of one of the deadliest spiders in the world.

A bite from an Australian funnel web spider can kill a human in 15 minutes, but a harmless ingredient found in the venom of one species can protect brain cells from being destroyed by a stroke, even when given hours after the event, scientists say.

If the compound fares well in human trials, it could become the first drug that doctors have to protect against the devastating loss of neurons that strokes can cause.

Researchers discovered the protective molecule by chance as they sequenced the DNA of toxins in the venom of the Darling Downs funnel web spider (Hadronyche infensa) that lives in Queensland and New South Wales.

Venom from three spiders was gathered for the study after scientists trapped and “milked exhaustively” three spiders on Orchid beach, about 400km north of Brisbane.

The molecule, called Hi1a, stood out because it looked like two copies of another brain cell-protecting chemical stitched together. It was so intriguing that scientists decided to synthesise the compound and test its powers. “It proved to be even more potent,” said Glenn King at the University of Queensland’s centre for pain research.

Strokes occur when blood flow to the brain is interrupted and the brain is starved of oxygen. About 85% of strokes are caused by blockages in blood vessels in the brain, with the rest due to bleeds when vessels rupture. Approximately six million people a year die from stroke, making it the second largest cause of death worldwide after heart attacks.

When a stroke happens, the oxygen level in the brain drops. This forces the brain to burn its primary fuel, glucose, very differently. Instead of oxidising glucose for energy, the brain switches to a process called anaerobic glycolysis. The reaction releases energy to keep the brain working, but it also produces acid, which can cause brain cells to die.

In a series of studies on rats, King showed that a single small dose of the spider venom molecule protected neurons from induced strokes. The compound works by blocking what are called ion channels in cells, specifically those that respond to the onset of acidic conditions in the brain.

Reporting in the journal, Proceedings of the National Academy of Sciences, King describes how administering Hi1a two hours after stroke reduced the extent of brain damage in rats by 80%. But the compound was still effective eight hours after stroke, reducing the amount of brain damage by about 65% when compared with untreated animals.

Rats that had the compound recovered far better than those that went without. “The untreated rats performed very badly after stroke. Their neurological and motor performance were terrible,” said King. But treatment with Hi1a “almost restored these functions to normal,” he added.

The researchers hope to start human trials of the compound in the next two years, but have more experiments to perform first. These will test whether the molecule works in all cases of stroke and is safe to use when blood vessels rupture in the brain, rather than become blocked. In the latest study, the compound was infused directly into the brain, but the scientists have found since that nasal deliver works too.

If trials show that the compound works, it could potentially transform the treatment of stroke patients. There are no drugs on the market that can protect the brain from stroke injuries. The best hospitals can offer are infusions of clot-busting drugs if a clot is to blame, or a surgical procedure called endovascular thrombectomy, which physically pulls the clot from the brain.

Before doctors can administer clot-busting drugs, they must confirm with a brain scan that the stroke was caused by a blockage. This is because the treatment thins the blood and could make matters worse if the stroke was caused by a haemorrhage. If Hi1a is found to be safe for people who have had with brain bleeds, it could be given to patients as soon as they reach a doctor. “The drug could be given in the ambulance to most stroke patients before hospital arrival, maximising the number of neurons that can be saved,” said King. “This should diminish the mortality from stroke and provide much better outcomes for those that survive as more brain function will be retained.”

Kate Holmes at the Stroke Association, said it was unknown whether Hi1a could be an effective treatment in humans. “We welcome any treatment that has the potential to reduce the damage caused by stroke, particularly if this can benefit people who are unable to arrive at hospital quickly,” she added. “Current treatments must be given in half this time period and it is too early for us to know if this research can offer an alternative for stroke patients.

“We urge for stroke to be treated as an emergency. The sooner a person can get to hospital after a stroke, the sooner the right treatment can be received which can improve survival and help recovery,” she said.

• This article was amended on 22 March 2017 to clarify that there are a several species of funnel web spiders.

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It's a fish eat tree world

It's a fish eat tree world | SciFrye | Scoop.it
An international team of scientists analyzed 147 northern lakes and found that many rely on nutrients from tree leaves, pine needles, and other land-grown plants to feed aquatic life. The study, published today in Science Advances, offers the most comprehensive analysis to-date on terrestrial subsidies to lake food webs.

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DNA Replication Errors Contribute to Cancer Risk 

DNA Replication Errors Contribute to Cancer Risk  | SciFrye | Scoop.it

A follow-up study confirms that random mutations acquired during normal stem cell division likely play a major role in cancer incidence.


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Ancient palace complex discovered in Mexican Valley of Oaxaca

Ancient palace complex discovered in Mexican Valley of Oaxaca | SciFrye | Scoop.it
A pair of archaeologists with the American Museum of Natural History has unearthed a palatial compound in El Palenque's plaza in the Oaxaca Valley in Mexico. In their paper published in Proceedings of the National Academy of Sciences, Elsa Redmond and Charles Spencer describe their work, what they have uncovered and how their findings fit with the emergence of organized states in Mesoamerica.

The Oaxaca Valley near the southern tip of Mexico has been offering up clues of past civilizations for several decades—a team has been working at the El Palenque site in particular since 1993. In this new effort, the researchers focused on a dig on the north end of the plaza—the site of what the researchers believe was the home and business center for the ruler of an ancient empire.

The palace has been dated to approximately 2,100 to 2,300 years ago, a time before the Aztecs. Most in the field believe that the civilization that existed in Oaxaca was among the earliest states to come into existence in Mesoamerica. Redmond and Spencer suggest that their findings at the palace site back up that theory.

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Live imaging of root–bacteria interactions in a microfluidics setup

Live imaging of root–bacteria interactions in a microfluidics setup | SciFrye | Scoop.it
Plant roots play a dominant role in shaping the rhizosphere, the environment in which interaction with diverse microorganisms occurs. Tracking the dynamics of root–microbe interactions at high spatial resolution is currently limited because of methodological intricacy. Here, we describe a microfluidics-based approach enabling direct imaging of root–bacteria interactions in real time. The microfluidic device, which we termed tracking root interactions system (TRIS), consists of nine independent chambers that can be monitored in parallel. The principal assay reported here monitors behavior of fluorescently labeled Bacillus subtilis as it colonizes the root of Arabidopsis thaliana within the TRIS device. Our results show a distinct chemotactic behavior of B. subtilis toward a particular root segment, which we identify as the root elongation zone, followed by rapid colonization of that same segment over the first 6 h of root–bacteria interaction. Using dual inoculation experiments, we further show active exclusion of Escherichia coli cells from the root surface after B. subtilis colonization, suggesting a possible protection mechanism against root pathogens. Furthermore, we assembled a double-channel TRIS device that allows simultaneous tracking of two root systems in one chamber and performed real-time monitoring of bacterial preference between WT and mutant root genotypes. Thus, the TRIS microfluidics device provides unique insights into the microscale microbial ecology of the complex root microenvironment and is, therefore, likely to enhance the current rate of discoveries in this momentous field of research.

Via Christophe Jacquet, Jean-Michel Ané
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Longer Telomeres Protect against Diseases of Aging: A Tale of Mice and Men

Longer Telomeres Protect against Diseases of Aging: A Tale of Mice and Men | SciFrye | Scoop.it

Gladstone scientists discovered how the ends of chromosomes protect mice from developing a human disease of aging.


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It is easier for a DNA knot… 

It is easier for a DNA knot…  | SciFrye | Scoop.it

Anyone who has been on a sailing boat knows that tying a knot is the best way to secure a rope to a hook and prevent its slippage. The same applies to sewing threads where knots are introduced to prevent them slipping through two pieces of fabric. How, then, can long DNA filaments, which have convoluted and highly knotted structure, manage to pass through the tiny pores of various biological systems? This is the fascinating question addressed by Antonio Suma and Cristian Micheletti, researchers at the International School for Advanced Studies (SISSA) in Trieste who used computer simulations to investigate the options available to the genetic material in such situations. The study has just been published in PNAS, the journal of the National Academy of Sciences of the Unites States.


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Scientists turn mammalian cells into complex biocomputers

Scientists turn mammalian cells into complex biocomputers | SciFrye | Scoop.it

Sophisticated genetic circuitry spliced into cells could lead to cancer therapies, tissue generation on demand


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Climate change puts invasive plants on the move

Climate change puts invasive plants on the move | SciFrye | Scoop.it
Climate change may force one of New England's invasive plant species to retreat north, while another will likely stay put and take over an even greater area, according to a new study by UConn faculty and former doctoral candidates.

Garlic mustard may disappear from the southern part of New England only to crop up in Canada, while Japanese barberry takes hold of the region.

The study of the two pervasive, widespread, and problematic invasive species was published in the Proceedings of the National Academy of Sciences, and provides evidence that climate change can both inhibit and facilitate invasive species growth. Few studies have linked plant demographics to climate change to understand invasive species.

Invasive species and climate change are two of the primary factors that alter ecological systems, but their interaction requires a greater understanding to improve the efficiency of management plans, according to the ecological and evolutionary biology team from the University of Connecticut, Yale University, the University at Buffalo, University of New Hampshire, and the Linda Loring Nature Foundation on Nantucket.

By tracking individual plants across environmental variations in temperature and precipitation, the researchers were able to predict their survival success under temperature changes predicted for 2041-2050. They compared those to the predictions for the species under current conditions.

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Reports that cancer is ‘mainly bad luck’ make a complicated story a bit too simple

Reports that cancer is ‘mainly bad luck’ make a complicated story a bit too simple | SciFrye | Scoop.it

New research predicts that 2 in 3 cancers are ‘due to bad luck’, we look at why the story is a bit more complicated than that.


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In a sample of blood, researchers probe for cancer clues

In a sample of blood, researchers probe for cancer clues | SciFrye | Scoop.it

UC Berkeley bioengineers have made an important step towards liquid biopsy technology


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Smartphone app could allow men to test their fertility at home

Smartphone app could allow men to test their fertility at home | SciFrye | Scoop.it
Gadget designed to clip onto a smartphone able to detect abnormal sperm samples with 98% accuracy in trials
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Purging the body of 'retired' cells could reverse ageing, study shows

Purging the body of 'retired' cells could reverse ageing, study shows | SciFrye | Scoop.it
Findings raise possibility that a future therapy that rids the body of senescent cells might protect against the ravages of old age
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Radical shakeup of dinosaur family tree points to unexpected Scottish origins

Radical shakeup of dinosaur family tree points to unexpected Scottish origins | SciFrye | Scoop.it
Cat-sized Scottish fossil proposed as candidate for common dinosaur ancestor in controversial study that could overthrow a century of dinosaur classification
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After the ISS: Big Plans for Commercial Space Habitats

After the ISS: Big Plans for Commercial Space Habitats | SciFrye | Scoop.it

Have you ever dreamt of living in outer space? Since the development of spaceflight, the off-Earth lifestyle has been limited to the select few aboard the International Space Station. For nearly 19 years, the station has played host to rotating teams of highly-trained international astronauts.

 

The ISS, however, may be nearing the end of its tenure in low-Earth orbit.1 While the U.S., Russia, Europe, Canada and Japan have all extended their involvement in ISS operations until 2024,2 the venerable space station may be facing decommission shortly thereafter. This doesn’t mean that we’re abandoning the idea of living in space—in fact, because of they way they are exiting, it signals precisely the opposite.

 

Soon, orbiting living spaces will no longer be the exclusive domain of agency-affiliated astronauts. Like so much in space, habitats are going commercial. By the 2020’s, NASA’s intention is to transition low-Earth orbit to the private sector—in terms of both supply and demand.3


Via Chuck Black, Dr. Stefan Gruenwald
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