SynBioFromLeukipposInstitute

Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

Just before his death in 1988, Richard Feynman wrote on his blackboard in his Caltech office, “What I cannot create, I do not understand.” This statement has lived on in a way that perhaps not even professor Feynman could have predicted: as a founding tenet of the field of synthetic biology.

Synthetic biology is defined as, “the design and engineering of biologically based parts, novel devices and systems as well as the redesign of existing, natural biological systems”1, and this emerging technology is rapidly changing the way industry and academia are approaching problems within a wide range of applications—particularly medicine and drug development.

Bioengineers now have a number of tools available to “create” biological systems from the bottom-up, which the experimenters, to some degree, are able to predict and control. To some extent, but not sufficiently; the complexities of seemingly simple systems can be daunting and challenging to overcome, with negative implications for synthetic biology technologies and products actually reaching the market.

The main issues are related to the vastness and complexity of biological systems. In order to develop biologically based circuits analogous to classical electronic circuits, the bioengineer is dependent on reliable parts with defined functions that can be connected to form higher-order, predictable networks. However, biomolecular components are often incompatible and only work within a certain pathway or network, and the system may be so overwhelmingly complex that the interactions between the parts cannot be entirely understood.

The bioengineer may start at an ostensibly simple component level, but as new pathways and interactions are constructed, he or she will need tools for rapid and comprehensive characterization of the system as a whole. Much work has been done to develop individual biomolecular components; however it is clear that there is a keenly felt need in the field of synthetic biology to be able to create designable and fully predictable interaction networks2,3.

In much the same way as is done for electronic systems and subsystems, each biobased pathway must be suitable for prototyping, individual optimization and rigorous testing. The design-build-test cycle for synthetic biology is therefore contingent upon the development of accurate models and robust algorithms for the components, subsystems and the whole system. Such models embody the “understanding” leading to the “creating”, according to the Feynman principle, and will be the key to integration of biological circuitry. Intrinsic to those models will be large amounts of experimental data, and here is where a major pinch point appears: the need for SCREAM technologies: small, cheap, rapid, extensible, automated, multiplexed technologies to enable detailed specification and characterization of the biological system at any point during development.

An illustration of this challenge is the development of aptamers using synthetic biology approaches. Aptamers are typically oligonucleotide molecules generated via systematic evolution by exponential enrichment (SELEX) and selected for high binding specificity to a certain target. Their unique stability and toxicity profiles, along with superior ligand-binding modifiability, have suggested they have great potential for use in diagnostics and therapeutics, especially as novel drug delivery platforms or drugs against previously intractable targets4-6. Aptamers also lend themselves very well as tools and components in drug development and synthetic biology networks.

For example, a bioengineer may make use of so-called riboswitches; RNA aptamers that are engineered to couple binding to a certain ligand with a conformation change that determines the output—such as expression of a gene or activation of a pathway— of a designed system. However, the SELEX method for screening a large (1014-1015) oligonucleotide library can be very laborious and fail to select an appropriate aptamer7. Therefore, technologies to extend the search space and making the process SCREAM, are in great demand in synthetic biology and drug development.

An example of a large-scale high-throughput experimental platform that will allow highly multiplexed screening of aptamer binding parameters (“binding screening”) is the use of high-resolution interferometry. Such platforms are designed to study,  rapidly and cheaply, surface-based interactions between biological molecules without the need to use elaborate labelling approaches. An interferometric, label-free approach allows a large number of binding experiments to be arranged in arrays and interrogated simultaneously; this provides deep multiplexing and highly-parallel operation to allow for large amounts of data to be collected in short time-frames. 

The incorporation of microfluidics to control the introduction of exogenous biochemical signals onto the arrays lends an additional dimension, allowing the combination of parts into small functional units, then into subsystems, interconnecting pathways and systems of increasing complexity. Platforms such as this, with appropriate sensitivity, flexibility and ease-of-use, open up opportunities for large coverage of interaction space in order to select the best aptamer.

The impact on aptamer development, and more generally synthetic biology, by the use of highly multiplex platforms may be significant, particularly in addressing problems regarding variable components, condition control and incompatibility. Multiplexed experimentation has the potential to open the door to massively parallel testing of biomolecular components for a multitude of parameters including binding kinetics and input/output robustness, allowing selection of appropriate parts for integrating into a designed system.

To return to the example of riboswitch aptamers, massive parallelization of interaction studies has the potential to generate detailed data on environmental parameters allowing precise sensing/response control; in addition, the array configuration of parts coupled with microfluidics may enable higher order network design by sequestering parts while providing absolute signal control.

Another considerable impact of the above technology on synthetic biology is that the high-fidelity, data-rich experiments will facilitate the construction of adaptive and predictive in silico network models.The use of large amounts of reproducible and experimental data will be crucial to quicken the pace of development of synthetic biology as a true engineering discipline.

The use of synthetic biology technologies in industrial applications is currently largely focused on innovation in medical devices (diagnostics and therapeutics) and energy production and storage (biofuels), but the potential to expand bioengineering across a wider range of scientific disciplines,  such as computing, smart materials and sensors, is under intense research.

The availability of suitable SCREAM technologies will be central for making synthetic biology real and for bringing synthetic biology products, such as novel drugs, to the market. Undoubtedly, as massively multiplex experimentation and development of predictive models continue to inform and shape our understanding of synthetic biological systems, the creating will ultimately be limited only by the bioengineer’s creativity. Indeed, to paraphrase Professor Feynman: “the more we understand, the more we will be able to create.” 

Prof. Sophia Roosth, history of science, Harvard University, spoke in Kaufmann Auditorium Thursday about the limits of the biological and how we define life in a new age of science, which defy typical expectations and definitions of life.

Karl Deisseroth, professor of bioengineering and of psychiatry and behavioral sciences at Stanford, won a $3 million 2016 Breakthrough Prize in life sciences for his contributions to the development of optogenetics, a technique that uses light to control the behavior of cells and has proved especially invaluable in the study of nerve-cell circuits in the brain.

Transcriptic has added CRISPR gene editing to the list of services provided by its robotic laboratory. The service expansion is being supported by Desktop Genetics, a developer of gene-editing software that is making its CRISPR library design algorithms available to Transcriptic.

Customers of Transcriptic can take advantage of the algorithms made available by Desktop Genetics by logging on to the web-based system that serves as the interface for all of the company's services. The site gives users control of experimental design parameters, allowing researchers to detail what they want to do and order Transcriptic's robots to carry out the assembly of their CRISPR libraries. Transcriptic and its partner think the combination will eliminate manual steps in the gene-editing process and in doing so save time and money. 

"Most labs are limited to an output of around eight cell lines per year," Desktop Genetics CEO Riley Doyle said in a statement. Teaming up with Transcriptic is intended to remove this limit and bring CRISPR to more researchers. "This partnership will allow our customers to scale their research by orders of magnitude by addressing several key bottlenecks, namely the enormous up-front capital expenditure required to set up high-throughput genome editing facilities, and an onerous CRISPR design process."

HERE ARE SOME words guaranteed to draw exasperated sighs among synthetic biologist: Food Babe. Greenpeace. Friends of the Earth. These are megaphone-wielding critics of genetically modified organisms. “They really have an agenda,” said a synthetic biology company rep, her eyes widening with concern, at Consumer Bio, a invite-only forum for venture capitalists and applied biology companies in San Francisco this week. Food Babe was most certainly not invited, but the anti-GMO message she’s perfected was the subtext of many conversations.

At Consumer Bio, companies both big and small dreamed about the potential of genetically engineering microbes to make food, perfumes, and new materials like lab-spun spider silk. The small companies included start-ups like the event’s sponsor, Ginkgo Bioworks, a Boston-based outfit that make flavors and fragrances with yeast. But the big players were there, too–Colgate, General Mills, L’Oreal. And most of them agreed that their biggest problem isn’t science. It’s branding.

Everyone there was afraid of “GMOs”—not the idea, but the word. “A big struggle everyone here has is how to do you talk about your product without calling it a genetically modified organism,” says Mark Bünger of the market research firm Lux Research. In a world where “is Monsanto” autocompletes to “evil” and “satan,” you want to stay far far away. But how do you tout the benefits of a technology when the well is already so poisoned?

One idea is you just forget about the technology—talk up the product instead. Unlike ag companies like Monsanto, most of these companies aren’t selling the actual genetically modified organism, but its product. Bolt Threads, for example, wants to make premium clothing from spider silk made by modified yeast. “It’s all about romance. It’s not about labs,” says Sue Levin, Bolt’s chief marketing officer. After she started the job last year, the company picked a new name—it used to be called “Refactored Materials”—and a sleek new logo.

Bolt will launch its first product next year, and Levin doesn’t plan to talk up “yeast” or “growth medium.” “I want the first thing people say about Bolt Threads to be ‘Oh my god, I want that,’” says Levin. Take the example of Tesla, which makes electric cars that are, importantly, flipping fantastic to drive.

But another tact is radical transparency, especially for more science-forward companies that want to allay the fears of consumers interested in the nitty gritty of genetic modification. Ginkgo Bioworks, which modifies yeast to make flavors like vanillin or scents like rose oil, is taking that road. “If you don’t talk about the process, people will be skeptical,” says Jason Kelly, a co-founder of the company. So Ginkgo has welcomed reporters, including WIRED’s (stay tuned), to its office in Boston, where big glass windows look into the gleaming white lab space.

Ginkgo also has an impressive scientific pedigree. Its founders include Tom Knight, a former MIT professor known as the father of synthetic biology, so the company has a stake in promoting the field. “About a year and a half ago, we decided we had to take the initiative,” says Kelly. And sponsoring events like Consumer Bio are part of it.

No matter how much you do or do not talk about the process, the imperative is to avoid the letters “GMO.” Revolution Bioengineering, which is genetically engineering a color-changing petunia, calls it “applied biology.” Ginkgo Bioworks talks about “designing” organisms to make “cultured ingredients.” Nobody’s hiding the actual process of their technology, but take away the emotionally charged letters of “GMO,” and Monsanto’s dark shadow no longer looms.

Rebranding GMO might just need to start with these smaller start-ups. The established companies at the meeting were more tempered in their enthusiasm. “I don’t think you can do it in an established brand,” says John Buckley of Kerry, an international food ingredients and flavors company. They’re happy to let startups take the risk—rather than risk the value of their established brand.

And that make sense for another reason way, too. When you hear about a big multinational corporation engineering microbes, that sounds like the plot for a dystopian video game. Monsanto couldn’t feed starving children without people casting aspersions. But a shiny Silicon Valley startup engineering microbes to change the world? That’s suddenly kind of exciting. Maybe even… disruptive…?

Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by clustered regularly interspaced short palindromic repeats (CRISPR)1, 2. Together with single-guide RNAs3, Cas9 also functions as a powerful genome engineering tool in plants and animals4, 5, 6, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications7, 8. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage9, 10, 11, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial protospacer adjacent motif (PAM) recognition12 and RNA–DNA base-pairing3 that serves as a final specificity checkpoint before DNA double-strand break formation.

NASA seems to believe that making space habitable will require more finesse than Elon Musk’s “let’s nuke Mars” plan, and has funded a couple of synbio projects which seek to provide “the means to produce food, medical supplies and building materials on site at distant destinations”. Dr Mark Blenner of Clemson University is working on recycling space waste (or ‘turning poop into food’) and explains that “it is impractical for astronauts to travel with all necessary supplies in future long-term space exploration missions…NASA has long been interested in converting space waste into useful molecules, such as fertilizer and propellant. These processes have typically focused on physical-chemical treatments. NASA’s recent interest in synthetic biology, in my opinion, is driven by the complex molecules that biochemistry can make, and by the wide variety of products that can be made.”

Although ideas such as terraforming are well known from scifi, NASA has long funded research such as that carried out by Prof. Robert Ferl and Dr. Anna-Lisa Paul at the University of Florida, who study the effects of space travel on gene expression in plants. Professor Ferl states “when we live on Mars we have to take our ecosystems and our agriculture with us”.

The Wilson Center's Synthetic Biology Project has released a short documentary on the growth of the do-it-yourself biology (DIYbio) movement as seen through a community DIYbio lab in Baltimore, Maryland.

The Rise of Do-It-Yourself Biology: A Look at the Baltimore Underground Science Space (BUGSS) explores the work of BUGSS, a fast-growing community lab on the east side of Baltimore, Maryland. BUGSS grew out of a group of interested students and professors at a local community college and now offers courses, lectures and the ability to experiment with biotechnology, from building microorganisms to modifying 3D printers.

In addition to providing an inside look at the BUGSS lab, the film explores the issues surrounding DIYbio community labs in general, including how they secure funding, where they find their equipment, and how they address concerns about biosafety.

"BUGSS is on the cutting edge of the DIYbio movement," says Dr. Todd Kuiken, senior program associate at the Synthetic Biology Project. "Spaces like BUGSS are providing creative and innovative outlets for scientists of all ages, while also providing educational opportunities where traditional academic institutions have lagged behind."

In 2013, the Synthetic Biology Project released Seven Myths and Realities about Do-It-Yourself Biology, a survey of DIYbio practitioners that challenges seven widely held beliefs about DIYbio, particularly that anonymous scientists are cooking up deadly epidemics in their basements. The report is available on the project's website: http://www.synbioproject.org/publications/6676/

The Rise of DIYbio was filmed on location at BUGSS in Baltimore and at the Wilson Center in Washington, DC. The film was directed and produced by Aaron Lovell, with the help of Joe Filvarof and Lee Gillenwater. David Rejeski, the director of the Science and Technology Innovation Program, is the film's executive producer. The Synthetic Biology Project is part of the Science and Technology Innovation Program at the Wilson Center.

The film can be found online here: http://www.synbioproject.org/news/project/bugss-doc/

The BUGSS website can be found here: http://www.bugssonline.org/

Synthetic biologist Josiah Zayner knows the perils of working alone. He’s spent the past two years as a research fellow in NASA’s synthetic biology program engineering bacteria that could someday terraform Mars, but the agency’s limited funding means that he has almost always worked alone. But Zayner wants to change that—he believes that more scientific minds and greater collaboration could yield biological solutions, such as new medical cures or ways to compost plastic, to some of the world’s most pressing problems. Now he has started a crowdfunding campaign on Indiegogo designed to bring the lab home for novice biohackers, teaching them to hack different organisms using the gene editing tool CRISPR.
Zayner’s company, The ODIN, offers several different kits designed to teach users the basics about biohacking equipment and processes so that they can start to innovate beyond it. Engineering kits range from $75 to $5,000 (the cheapest one containing CRISPR is $130), and each contains a micropipette, pipette tips, a microcentrifuge rack and tubes, and petri dishes with growing media. Hackers can choose whether they want to edit prokaryotes (such as bacteria) or eukaryotes (yeast), and the kits will contain step-by-step written and video instructions for how to edit the genes of the organism. Though The ODIN already sells a few different kits, the ones offered through the Indiegogo campaign are the only ones designed for biohackers and to use CRISPR.

The advent of potent genome engineering techniques and efficient DNA assembly methods has greatly expanded our capacities to modify genomes and have fueled metabolic engineering and synthetic biology endeavours. Here we provide an overview on genome engineering methods ranging from non-programmable tools such as site-specific recombinases to highly specific, programmable strategies as CRISPR–Cas9. Advantages and limitations of the methods for the generation of custom-designed organisms are compared. Certain methods such as group II introns and recombineering are limited to prokaryotes, whereas zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and CRISPR–Cas9 are universally applicable for all domains of life. We summarize also popular DNA assembly methods, suitable for generating multigene pathways for metabolic engineering and artificial constructs for synthetic biology or entire genomes.

When your company builds custom organisms, you need a lot of DNA — millions of base pairs, to be exact.

Boston-based Ginkgo Bioworks has announced plans to buy 100 million base pairs of synthetic DNA, an order the company says is 10 percent of all synthetic DNA that will be made in 2015 and 20 times the amount of synthetic DNA Ginkgo received this year.

“We’ll get in probably 20,000 tubes that are filled with genetic material,” said Jason Kelly, chief executive of Ginkgo. “It’s an enormous order.”

Ginkgo needs so much custom DNA, Kelly said, because of the increasing popularity of its method of creating custom organisms. The agreement to buy the DNA is a commitment to order the base pairs over the next year for Ginkgo’s various customers.

The company designs and manufactures custom yeasts that, when brewed like beer, make something far more interesting such as synthetic rose oil. An automated manufacturing process at Ginkgo’s offices has made it easier and quicker to create custom yeasts.

“What we’ve done is we’ve taken those processes that are historically done by PhDs in a bench and we’ve automated them,” Kelly said.

Much of Ginkgo’s process is dependent on a custom-built foundry that does much of the work of putting the organisms together.

But in order for this to work, Ginkgo has to identify the genetic sequence responsible for producing rose oil in rose petals. Then, the company places orders for synthesized versions of that DNA to insert into yeast.

“Biological research and development is a time-intensive, sequence- and gene-specific effort that has been constrained by relatively limited DNA,” said Emily Leproust, chief executive of Twist Bioscience in a statement.

Under a partnership with French fragrance maker Robertet, Ginkgo developed yeast that emits rose oil for perfumes. Last week, Ginkgo said it had expanded the partnership to include producing coconut, peach, apricot and mango scents.

Synthetic biology has taken off in recent years. Companies have raised hundreds of millions of dollars from investors, including more than $50 million that went to Ginkgo.

“The field of synthetic biology is all about making biology easier to engineer,” said John Cumbers, founder of synbiobeta, a trade group for the industry. Cumbers said the idea of engineering biology shouldn’t sound ominous, and could lead to improvements for fuels and energy production and other chemicals.

Synthetic DNA is made by gluing together the chemicals that make up DNA in our bodies to construct a custom genetic code. That process — and the rest of the synthetic biology field — is very much in its early days.

“We’re back in the day of linear programming and mainframe computers and using punch cards,” Cumbers said.

Highlights
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Synthetic biology at the cellular and ecological level holds significant promise to solve imminent global challenges in agriculture, energy and human health
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To achieve these goals, engineered biological solutions will need to be designed effectively to survive, grow and function in complex environments beyond the laboratory
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The engineering design space contains trade-offs among the resource limitations to support cellular fitness, implementation of a desired function and mechanisms to constrain the organism to a desired niche
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Natural biological systems employ multiple strategies at the cellular, population and microbial community levels to continuously adapt to changing environments and to achieve a stable ecological function
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Engineering efforts should aim to extract natural design principles to develop compact and efficient designs that implement functionally equivalent behaviors

We may think of our fragrances today as delicate chemical concoctions, divided from the erratic smells of the world by the glass sheaths of over-contemplated bottles. But the first fragrances were not only chemical, they were biological, and made directly from things in the wild and smelly environment, like animal excretions.

Now, a synthetic biology foundry in Boston, Ginkgo Bioworks, which has dubbed itself as “the organism company,” is re-wilding our noses with raw biological smells by drilling right down into an organism’s DNA.

In a room in Times Square packed with people who want to #growthefuture by turning the biology of microbes into assembly lines that manufacture useful things for us, the idea that we can, and should, biofabricate whatever we want from “living factories” was buzzing at last months Biofabricate 2015 conference. This is where Christina Agapakis, the creative director of Ginkgo Bioworks, took the stage to explain how a rose is not really a regular rose any longer with Ginko’s futuristic perfuming methods.

The company is never going to bottle the exact natural rose smell you know, and it doesn’t want to. Instead, Ginkgo Bioworks wants to extend it towards new varieties of rose smells that its scientists can build up, bit by bit, with chunks of DNA.