Tardigrade protein helps human DNA withstand radiation.

Tardigrade protein helps human DNA withstand radiation.

Eye of Science/Science Photo Library  Experiments show that the tardigrade’s resilience can be transferred to cultures of human cells.

Water bears are renowned for their ability to withstand extreme conditions.
Tardigrades, or water bears, are pudgy, microscopic animals that look like a cross between a caterpillar and a naked mole rat. These aquatic invertebrates are consummate survivors, capable of withstanding a host of extremes, including near total dehydration and the insults of space.

Now, a paper1 published on 20 September in Nature Communications pinpoints the source of yet another tardigrade superpower: a protective protein that provides resistance to damaging X-rays. And researchers were able to transfer that resistance to human cells.

“Tolerance against X-ray is thought to be a side-product of [the] animal's adaption to severe dehydration,” says lead study author Takekazu Kunieda, a molecular biologist at the University of Tokyo. According to Kunieda, severe dehydration wreaks havoc on the molecules in living things. It can even tear apart DNA, much like X-rays can.

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The researchers wanted to know how tardigrades protected themselves against such harsh conditions. So Kunieda and his colleagues began by sequencing the genome of Ramazzottius varieornatus, a species that is particularly stress tolerant. It's easier to study processes within the tardigrade's cells when the animal's genome is inserted into mammalian cells, says Kunieda. So researchers manipulated cultures of human cells to produce pieces of the water bear's inner machinery to determine which parts were actually giving the animals their resistance.

Eventually, Kunieda and his colleagues discovered that a protein known as Dsup prevented the animal's DNA from breaking under the stress of radiation and desiccation. And they also found that the tardigrade-tinged human cells were able to suppress X-ray induced damage by about 40%.

Genomic treasure trove

“Protection and repair of DNA is a fundamental component of all cells and a central aspect in many human diseases, including cancer and ageing,” says Ingemar Jönsson, an evolutionary ecologist who studies tardigrades at Kristianstad University in Sweden.

This makes the new paper’s findings “highly interesting for medicine”, says Jönsson. It opens up the possibility of improving the stress resistance of human cells, which could one day benefit people undergoing radiation therapies.

Kunieda adds that these findings may one day protect workers from radiation in nuclear facilities or possibly help us to grow crops in extreme environments, such as the ones found on Mars.

Bob Goldstein, a biologist at the University of North Carolina at Chapel Hill who helped to sequence the genome of another tardigrade species2, says the research is exciting and clever. He also thinks that the study’s authors are correct in predicting that this is probably just the first of many such discoveries.

“The tardigrade is resistant to a lot of different kinds of extremes,” says Goldstein. And this means that the animals must have many different ways of protecting themselves.

“We are really just at the beginning of exploring the genetic treasure that the tardigrade genome represents,” says Jönsson.

Nature doi:10.1038/nature.2016.20648

References

Hashimoto, T. et al. Nature Commun. http://dx.doi.org/10.1038/ncomms12808 (2016).
Boothby, T. C. et al. Proc. Natl Acad. Sci. USA 112, 15976–15981 (2015).


ORIGINAL: Nature

By Jason Bittel
20 September 2016

 

Bitter fight over CRISPR patent heats up

Unusual battle among academic institutions holds key to gene-editing tool’s future use.

Steve Jennings/Getty/Breakthrough Prize.Biologist Jennifer Doudna of the University of California, Berkeley, helped to develop the CRISPR gene-editing system.

A versatile technique for editing genomes has been called the biggest biotechnology advancesince the polymerase chain reaction (PCR), and the US Patent and Trademark Office (USPTO) is set to determine who will reap the rewards.

On 11 January, the USPTO granted a request to review a key patent awarded for the technique, known as CRISPR–Cas9. The outcome of the ensuing proceedings, called a patent interference, could be worth millions to the research institutions that are at war over the relevant patents. It might also influence who is allowed to use the technology — and under what terms.

“This is an absolutely humungous biotech patent dispute,” says legal scholar Jacob Sherkow of New York Law School. “We’re all waiting with bated breath.”

CRISPR–Cas9 is a bacterial defence system that uses the enzyme Cas9 to snip DNA at sites determined by the sequence of a ‘guide’ strand of RNA. Scientists can disable, replace or tweak genes by using the technique to rewrite snippets of DNA sequences. Use of the technology in research has exploded, thanks to CRISPR–Cas9’s relative simplicity and versatility compared to other gene-editing methods. Several companies have sprung up to harness the technique for generating improved crops, research reagents and therapies for human genetic diseases.

The roots of the CRISPR–Cas9 dispute date back to 2012, when researchers reported that they had reprogrammed the system to cut strands of isolated DNA at sites of their choosing1. The team, led by biologists Jennifer Doudna at the University of California, Berkeley, and Emmanuelle Charpentier, now at the Max Planck Institute for Infection Biology in Berlin and Umeå University in Sweden, filed a patent application on 15 March 2013.

By then, publications had emerged from other groups showing that the method works in human cells2–4 and bolstering dreams of CRISPR-based gene therapies — the basis for several companies that have sprung up to capitalize on the technique.

One of those groups, led by synthetic biologist Feng Zhang of the Broad Institute and the Massachusetts Institute of Technology, both in Cambridge, filed a patent application for the CRISPR–Cas9 technique in October 2013. The institutions filed the patent under a special expedited review programme, and it was granted in April 2014. Zhang has since been awarded additional patents on the technology. The original Doudna–Charpentier patent remains under review.

A pitched battle

In April 2015, the Berkeley team asked the USPTO to begin an interference proceeding to determine which team was the first to invent the technique. The proceedings will be much like a court case, with both sides presenting evidence culled from publications and laboratory notebooks. “Once the [USPTO] declares an interference, that’s really when the fur is going to fly,” Sherkow predicted in a June interview.

Nature special:CRISPR — the good, the bad and the unknown

The patent interference is also a testament to the high stakes involved: companies aiming to use CRISPR–Cas9 for gene therapy have raised hundreds of millions in venture capital and other funds in under three years. One company, Editas Medicine in Cambridge, Massachusetts, has already filed to go public.

Arti Rai, a legal scholar at Duke University in Durham, North Carolina, says that it is unusual for academic research institutions to battle so intensely over a patent. Instead, such institutions usually come to an agreement to share rights to the invention. “This seems more bitter than disputes I’ve heard of in the past,” she adds.

The two patents in question make broad claims to 'foundational' intellectual property thought to be necessary for most lucrative CRISPR–Cas9 applications. But many patents have been filed on CRISPR–Cas9 technologies, and there is still the chance that the winner of the interference will face additional challenges in court. Zhang's group has also reported another enzyme, called Cpf1, that could provide an alternative to Cas9. Researchers expect other alternatives to emerge with time.

As for the various CRISPR–Cas9 companies, Zhang remains involved in Editas, which was founded by both Zhang and Doudna, among others, in 2013. Doudna has since severed ties with Editas and thrown her support behind Intellia Therapeutics, also in Cambridge. Charpentier, meanwhile, co-founded CRISPR Therapeutics of Basel, Switzerland.

Licensing looms

For now, it is unclear how the dispute will affect researchers who use CRISPR–Cas9, if it does so at all. Academics who might use the technology for basic research make unattractive targets for patent lawsuits, says Rodney Sparks, a biotechnology patent counsel at the University of Virginia in Charlottesville. “Patent holders might send out a few cease-and-desist letters, but they probably won’t sue academic researchers,” he says. Doing so would take time and money with little reward: the spoils in a patent lawsuit are typically damages or a share of royalties from a marketed product. That leaves little to gain from suing academics who are not selling anything. But those who intend to use their research as the basis for a start-up company will need to be wary, Sparks says.

Some patent holders do ask that even scientists doing basic research take out a licence on a patented technology, typically for a fairly small fee. Such was the case for PCR, says Warren Woessner, a lawyer at Schwegman Lundberg and Woessner in Minneapolis, Minnesota. Woessner recalls how, during his previous career as a scientist, his institution decided to patent a method he developed. Officials at the institution later noticed that someone had published a paper that used the technique without a licence. “They sent the professor a little note,” recalls Woessner.“‘We have a patent on this. Pay up.’” The professor did.

The Broad Institute has noted on its website that it will continue to make CRISPR–Cas9 reagents available to the community, and has given no indication that it will pursue licensing fees from academics. But Sherkow warns against assuming that the spirit of academic camaraderie will prevail: licensing revenue has become increasingly important, particularly for major research institutions, he says. “We’re just living in a brave new world these days.”
Nature doi:10.1038/nature.2015.17961

ORIGINAL: Nature

Heidi Ledford

12 January 2016

DNA Manufacturing Enters the Age of Mass Production

Synthetic-biology startups adopt technologies from the computer industry

Illustration: Elias Stein

Emily Leproust, CEO and cofounder of the buzzy biotech startup Twist Bioscience, is an industrialist on the nanoscale. “I remind everyone at Twist, we are a manufacturing company,” she says. “We manufacture DNA.”

Photo: Twist Bioscience
DNA Factory: Twist Bioscience’s machine
builds DNA strands inside
600-nanometer wells on a silicon plate.

Twist is part of the young industry of synthetic biology, in which living organisms are the product and a biology lab is the factory floor. By manufacturing strands of DNA—assembling the genetic code of life from its basic components—scientists are creating organisms the likes of which the world has never seen. And these new life forms can be decidedly useful: Biologists have produced yeast cells that excrete pharmaceuticals and algae that brew jet fuel.

This burgeoning business sector has been hampered by the labor-intensive nature of DNA assembly, a painstaking process requiring trained personnel. Now, nimble startups are competing to fashion automated DNA assembly lines that would make Henry Ford proud, using techniques copied from the fabs that make computer chips. As their innovations bring down the cost of constructing DNA strands, these entrepreneurs are aiming for a low price point, which they say will cause a market boom. Twist Bioscience, which will begin commercial operations at its San Francisco headquarters in 2016, is a leading contender in that race to the bottom.

Genetic material is composed of molecules called nucleobases; the four types of bases in DNA are identified by the letters A, C, G, and T. The order of these letters serves as a code that instructs an organism how to build its cells and carry on the functions of life. In human beings, this code is about 3.2 billion letters long, while the yeast used in baking and beer brewing has a code of about 12 million letters. If you tweak the order of the letters, you tweak the organism’s instructions. Synthetic biologists have written new snippets of code and inserted them into yeast DNA, causing the microbe to churn out, for example, the omega-3 fatty acids found in fish oil supplements or the aromatic oils normally produced by roses.

Matter of Fact

Mycoplasma laboratorium: The name given to the first “synthetic organism,” a bacterium whose 1-million-base genome was assembled from scratch.

Constructing a strand of DNA isn’t complicated; in fact it’s a routine procedure performed in labs all over the world. But that procedure is typically carried out by hand, says Twist’s Leproust: “Microbiology is manual labor. You have a Ph.D. student moving liquid from one test tube to the next all day long.” So she and her cofounders invented a machine that automates the construction process.

The heart of the machine is a silicon plate pocked with 10,000 tiny wells, which are etched using the same photolithography techniques perfected by computer chip manufacturers. A different strand of DNA can be constructed in each 600-nanometerwide well. The machine does “the exact same chemistry” as a Ph.D. student would do, Leproust says, “only in a volume that’s 100 times smaller.”

Twist isn’t selling its machine but rather its DNA manufacturing services, which are aimed at researchers and startups seeking new genetic modifications that might prove useful. In 2015 the company began production runs for select customers; 2016 will see Twist’s full commercial launch. DNA assembly is priced on a cost-per-base model, and Leproust says her company’s 10-cents-per-base starting price is already the best in the industry. But she’s aiming for a 2-cent price point: “That’s the point at which researchers can significantly scale experiments and will no longer be limited by the cost of DNA,” she says. Today, customers typically order DNA strands of 300 to 1,800 bases in length, Leproust says.

1,600 Bases: Length of gene for insulin (INS)

81,000 Bases: Length of gene for breast cancer risk (BRCA1)

Another synthetic-biology startup in the San Francisco area, Zymergen ("ROBOTICS FOR HIGH THROUGHPUT BIOLOGY"), offers customers a broader set of services. The company not only 

• constructs DNA snippets on the cheap, it also 
• inserts that DNA into microbes and 
• monitors the outcome. 

Chief science officer Zach Serber explains that the results can inform the next round of DNA design, letting customers iterate quickly as they look for their ideal organism. “You cast a wide net,” Serber says, “and when you find a variation that improves the microbe’s performance, then you double down.”

Such setups have led to excited talk of a synthetic-biology industry based on “organism fabs.” But the promise of mass-produced DNA doesn’t impress Rob Carlson, a biotech consultant and managing director of the BioEconomy Capital venture fund. “I don’t understand the business model,” he says.

Carlson is skeptical that cheap DNA assembly will lead to a proliferation of startups with ideas for profitable microbes. “So you can make and test a whole bunch more DNA—but that’s not the hard part,” he argues. “Going from test tube to bench scale to commercial scale, that’s 90 percent of cost.” For a startup to build a business around a yeast that cranks out a pharmaceutical, for example, it must manage massive tanks full of microbes. Reducing the cost of the initial DNA manufacturing would only give the company pocket money, Carlson says: “Hooray, they get to buy beer, or more pizza on Friday.”

ORIGINAL: IEEE Spectrum

By Eliza Strickland

23 Dec 2015

MIT, Broad scientists overcome key CRISPR-Cas9 genome editing hurdle

Courtesy of Ian Slaymaker/Broad Institute of MIT and Harvard

The researchers used structural knowledge of Cas9 to guide engineering of a highly specific genome-editing tool.

MIT, Broad scientists overcome key CRISPR-Cas9 genome editing hurdle

Team re-engineers system to dramatically cut down on editing errors; improvements advance future human applications.

The following is adapted from a press release issued today by the Broad Institute.

Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have engineered changes to the revolutionary CRISPR-Cas9 genome editing system that significantly cut down on “off-target” editing errors. The refined technique addresses one of the major technical issues in the use of genome editing.

The CRISPR-Cas9 system works by making a precisely targeted modification in a cell's DNA. The protein Cas9 alters the DNA at a location that is specified by a short RNA whose sequence matches that of the target site. While Cas9 is known to be highly efficient at cutting its target site, a major drawback of the system has been that, once inside a cell, it can bind to and cut additional sites that are not targeted. This has the potential to produce undesired edits that can alter gene expression or knock a gene out entirely, which might lead to the development of cancer or other problems. 

In a paper published today in Science, Feng Zhang and his colleagues report that changing three of the approximately 1,400 amino acids that make up the Cas9 enzyme from S. pyogenes dramatically reduced “off-target editing” to undetectable levels in the specific cases examined. Zhang is the W.M. Keck Career Development Professor in Biomedical Engineering in MIT’s departments of Brain and Cognitive Sciences and Biological Engineering, and a member of both the Broad Institute and McGovern Institute.

Zhang and his colleagues used knowledge about the structure of the Cas9 protein to decrease off-target cutting. DNA, which is negatively charged, binds to a groove in the Cas9 protein that is positively charged. Knowing the structure, the scientists were able to predict that replacing some of the positively charged amino acids with neutral ones would decrease the binding of “off target” sequences much more than “on target” sequences.

After experimenting with various possible changes, Zhang’s team found that mutations in three amino acids dramatically reduced “off-target” cuts. For the guide RNAs tested, “off-target” cutting was so low as to be undetectable.

The newly-engineered enzyme, which the team calls “enhanced” S. pyogenes Cas9, or eSpCas9, will be useful for genome editing applications that require a high level of specificity. The Zhang Lab is immediately making the eSpCas9 enzyme available for researchers worldwide. The team believes the same charge-changing approach will work with other recently described RNA-guided DNA targeting enzymes, including Cpf1, C2C1, and C2C3, which Zhang and his collaborators reported on earlier this year.

The prospect of rapid and efficient genome editing raises many ethical and societal concerns, says Zhang, who is speaking this morning at the International Summit on Gene Editing in Washington. “Many of the safety concerns are related to off-target effects,” he says. “We hope the development of eSpCas9 will help address some of those concerns, but we certainly don’t see this as a magic bullet. The field is advancing at a rapid pace, and there is still a lot to learn before we can consider applying this technology for clinical use.”

ORIGINAL: MIT News

News Office 

December 1, 2015