Monthly Archives: February 2017

February 9, 2017 by Kathryne Metcalf Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research, however have limited sequence specificity and availability. The Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) is capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length. Credit: Behnam Enghiad and Huimin Zhao, University of Illinois at UrbanaChampaign

Research by Professor of Chemical and Biomolecular Engineering Huimin Zhao and graduate student Behnam Enghiad at the University of Illinois is pioneering a new method of genetic engineering for basic and applied biological research and medicine. Their work, reported in ACS Synthetic Biology on February 6, has the potential to open new doors in genomic research by improving the precision and adherence of sliced DNA.

"Using our technology, we can create highly active artificial restriction enzymes with virtually any sequence specificity and defined sticky ends of varying length," said Zhao, who leads a synthetic biology research group at the Carl R. Woese Institute for Genomic Biology at Illinois. "This is a rare example in biotechnology where a desired biological function or reagent can be readily and precisely designed in a rational manner."

Restriction enzymes are an important tool in genomic research: by cutting DNA at a specific site, they create a space wherein foreign DNA can be introduced for gene-editing purposes. This process is not only achieved by naturally-occurring restriction enzymes; other artificial restriction enzymes, or AREs, have risen to prominence in recent years. CRISPR-Cas9, a bacterial immune system used for "cut-and-paste" gene editing, and TALENs, modified restriction enzymes, are two popular examples of such techniques.

Though useful in genetic engineering, no AREs generate defined "sticky ends"an uneven break in the DNA ladder-structure that leaves complementary overhangs, improving adhesion when introducing new DNA. "If you can cleave two different DNA samples with the same restriction enzyme, the sticky ends that are generated are complementary," explained Enghiad. "They will hybridize with each other, and if you use a ligase, you can stick them together."

However, restriction enzymes themselves have a critical drawback: the recognition sequence which prompts them to cut is very shortusually only four to eight base pairs. Because the enzymes will cut anywhere that sequence appears, researchers rely on finding a restriction enzyme whose cut site appears only once in the genome of their organism or plasmidan often difficult proposition when the DNA at hand might be thousands of base pairs long.

This problem has been partially solved simply by the sheer number of restriction enzymes discovered: more than 3600 have been characterized, and over 250 are commercially available. "Just in our freezer, for our other research, we have probably over 100 different restriction enzymes," said Enghiad. "We look through them all whenever we want to assemble something ... the chance of finding the unique restriction site is so low.

"Our new technology unifies all of those restriction enzymes into a single system consisting of one protein and two DNA guides. Not only have you replaced them, but you can now target sites that no available restriction enzymes can."

Enghiad and Zhao's new technique creates AREs through the use of an Argonaute protein (PfAgo) taken from Pyrococcus furiosus, an archeal species. Led by a DNA guide, PfAgo is able to recognize much longer sequences when finding its cut site, increasing specificity and removing much of the obstacles posed by restriction enzymes. Further, PfAgo can create longer sticky ends than even restriction enzymes, a substantial benefit as compared to other AREs.

"When we started, I was inspired by a paper about a related proteinTtAgo. It could use a DNA guide to cleave DNA, but only up to 70 degrees," explained Enghiad. "DNA strands start to separate over 75 degrees, which could allow a protein to create sticky ends. If there were a protein that was active at higher temperatures, I reasoned, that protein could be used as an artificial restriction enzyme.

"So I started looking for that, and what I found was PfAgo."

In addition to replacing restriction enzymes in genetic engineering processes, Enghiad and Zhao believe their technology will have broad applications in the biological research. By creating arbitrary sticky ends, PfAgo could make assembly of large DNA molecules easier, and enables cloning of large DNA molecules such as biochemical pathways and large genes.

The application of these techniques is broad-reaching: ranging from discovery of new small molecule drugs to engineering of microbial cell factories for synthesis of fuels and chemicals to molecular diagnostics of genetic diseases and pathogens, which are the areas Zhao and Enghiad are currently exploring.

"Due to its unprecedented simplicity and programmability (a single protein plus DNA guides for targeting), as well as accessibility ... we expect PfAgo-based AREs will become a powerful and indispensable tool in all restriction enzyme or nuclease-enabled biotechnological applications and fundamental biological research," said Zhao. "It is to molecular biology as the CRISPR technology is to cell biology."

Explore further: Prevention of RNA virus replication

More information: Behnam Enghiad et al, Programmable DNA-Guided Artificial Restriction Enzymes, ACS Synthetic Biology (2017). DOI: 10.1021/acssynbio.6b00324

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New method of genetic engineering indispensable tool in ... - Phys.org - Phys.Org

Over the last few years, advances in science have made the kind of experiments once only accessible to PhDs with fancy labs far more attainable. College undergrads are constructing gene drives. Anyone can buy a kit on the internet to concoct their own bioluminescent beer.

The German government, it seems, is none too pleased with this development. Two weeks ago its consumer protection office issued a statement making clear just how upset it is: Any science enthusiast doing genetic engineering outside of a licensed facility, it wrote, might face a fine of 50,000 or up to three years in prison.

The statement sent a wave of shock through the DIY bio community.

This is the first time Ive ever heard of a government calling out the DIY community specifically, said Todd Kuiken, a senior research scholar with the Genetic Engineering and Society Center at North Carolina State University.

The law behind the German DIY bio crackdown isnt new. The government was simply reminding so-called biohackers of a long-existing law that forbids genetic engineering experiments outside of laboratories supervised and licensed by the state.

But there is concern over how the pledge to enforce those rules may stymy the growth of the DIY science movement, and whether Germanys statement may inspire other European nations to take a similarly firm stance.

I am worried that the mentality could spread to other countries, said Josiah Zayner, who runs The Odin, a US company that sells DIY CRISPR kits.

Europe is generally much stricter in its regulation of genetic engineering and genetically modified products than the United States. In some countries, it is unclear whether DIY genetic engineering is legal at all.

A spokesperson for Germanys consumer protection office, the BVL, told Gizmodo that officials gathered in November to discuss concerns over the appearance of cheap DIY genetic engineering kits for sale on the internet, and decided it should issue a warning. Companies like The Odin and Amino Labs sell kits that make experimenting with DNA not much more difficult that whipping up a batch of brownies with a box of mix. Amino Labs compact, table-top bacteria lab is even sort of reminiscent of an Easybake oven, with its bright colors and playful name, the DNA Playground.

The statement has to be seen in light of the newly formed DIY biology scene and due to the appearance of low-priced DIY biology kits in online shops, the BVL told Gizmodo, via email.

At the moment, the BVL said it has not used the law to bring any criminal chargers against biohackers, though it may do so in the future.

Its difficult, but not impossible, for an individual in Germany to receive explicit government permission to do genetic engineering experiments outside of a lab. In Ireland, a PhD dropout named Cathal Garvey won such approval from the Irish government back in 2012.

Im pretty sure that laws will prohibit me from continuing my research at a later state, said Bruno Lederer, a German biohacker who hopes that loopholes in the law will allow his work to continue for now. I think its a shame that Id have to do illegal things in order to do independent research.

The BVL conceded that the new rules will make it virtually impossible for a lone scientist to meet the legal requirements to do genetic engineering. To begin with, any lab needs a project manager qualified by academic credentials such as a masters degree in science. Labs also require a commissioner for biological safety who is similarly qualified.

This makes genetic engineering experiments rather unattractive for individuals, the BVLs spokesman said.

Community biology labs, which often receive oversight and advisement from traditional scientists, shouldnt have an issue getting licensed. But not every DIY scientist lives near or has the resources to join a community lab. If the DIY bio movement is about making science accessible to those outside the Ivory Tower of academia, the German governments statement represents a serious roadblock.

If you are not living in a big city, access to a community biolab or an informal learning environment like a maker space is difficult, said Orkan Telhan, whose company, Biorealize, is in the process of developing its own DIY bio kits. There is no doubt that the field has to be regulated to mitigate adverse outcomes, but we need alternative ways to engage new audiences with biology.

In the US, biohackers operate in more of a regulatory gray areaoften regulations do not apply to them simply because no one ever conceived that self-taught scientists would one day pursue sophisticated biology experiments in their garages. But as the DIY community here has grown and sought to not just experiment at home, but sell its creations to the public, it, too, has increasingly faced regulatory run ins. In the US, DIY scientists are subject to the same rules as any other scientists. As in Germany, those regulations can be difficult to comply for an individual to comply with.

I dont think its entirely uncalled for to evaluate some of these spaces like community labs to make sure that they are operating in a safe manner, said Kuiken. I think that they are already operating safely, but currently there is no system in the US to determine that.

Germanys statement does offer one silver lining: it offers the rare clear guidance for what rules biohackers must comply with in order to go about their work legally.

Germany stating their position is a step forward in clarification, said Julie Legault of Amino Labs. Hopefully other countries will clarify their own rules as well.

The only question now is whether those rules will prevent biohackers from continuing with their work at all.

Read more from the original source:
Germany Is Threatening Biohackers With Prison - Gizmodo