Impossible Foods and Other Achievements of Pat O. Brown

  • Brown’s Microarray Publications Started a Revolution in DNA/RNA Analysis
  • Open Access Publishing Was an Unintended Consequence of His Microarray Research
  • Brown’s Passion for Bettering Earth Led to Invention of the Plant-Based Impossible Burger

Impossible Foods is a company founded by Patrick “Pat” O. Brown that wants to transform the global food system by inventing foods we love, without compromise. It’s first commercial product uses 0% meat and 100% plants to recreate everything—i.e. sights, sounds, aromas, textures and flavors—of a big, juicy burger, aptly named the Impossible Burger. “Impossible” because this was not thought to be doable, as many “veggie” burgers have fallen short on appearance, texture and—importantly—taste.

But before I tell you more about the circumstances and science of this game changer of a burger by Brown and company, let me start with his background as related to nucleic acid research, specifically microarrays.

Pat O. Brown and Microarrays

Pat O. Brown. Taken from Wikipedia.org

Brown received his BS, MD, and PhD degrees all from the University of Chicago, where he worked and published with Nicholas R, Cozzarelli on topoisomerases, which are enzymes that participate in the overwinding or underwinding of DNA. Brown did his postdoctoral research with uber-famous Nobel Laureates J. Michael Bishop and Harold Varmus at the University of California, San Francisco.

Brown went on to become a professor at Stanford University and in 1995 was the first to report (along with his colleagues) the use of microarrays for high-throughput analysis of nucleic acid. This seminal article published in venerable Science magazine and titled Quantitative monitoring of gene expression patterns with a complementary DNA microarray has now been cited more than 11,000 times in Google Scholar.

This publication described general methods for attaching cDNA probes for genes of interest onto glass microscope slides using a high-speed arraying machine (aka robotic printing). These were then hybridized to fluorescently labeled cDNA derived from mRNA by reverse transcription with dNTPs including labeled dCTP akin to dye labeled dNTPs offered by TriLink for such applications. Slides were then fluorescently scanned to obtain “spots” having pseudo-color intensities for quantitation relative to a “spike in” reference gene, as shown below.

Taken from Brown & coworkers Science (1995).

This paper triggered the genesis of what would become a highly competitive microarray industry, which I think of as going from “seeing spots to seeing dollars.” Interested readers can find much information about this in a review by pioneering experts during that time. A brief synopsis of this commercialization involving Brown is as follows.

From Microarrays to Open Access

Taken from plos.org

During his time at Stanford, Brown and his coworkers were using microarrays to generate huge amounts of data on gene expression profiling that required detailed analysis of even larger amounts of information previously published in many different journals. Although many of these journals were available online via a subscription, others were not, and almost all strictly forbade downloading and automated analysis. This thwarted Brown and others from compiling databases for anyone to use as needed. In other words, it prevented Open Access—allowing everyone, everywhere to have unrestricted, free access to this information.

Brown mulled over various ways for researchers to share their data, and in a coffee shop discussion with Harold Varmus, who was then Director of the NIH, they agreed on the possibility of a NIH-hosted computer server where scientists could post their work, and where it would be organized in a systematic way. Shortly thereafter in 1999, Varmus posted on the Director’s website a draft proposal for something that was dubbed e-Biomed.

In 2001, Brown helped lead the Public Library of Science (PLOS) initiative to make published scientific research open access and freely available to researchers in the scientific community. PLOS quickly grew in popularity, as have other Open Access journals, and PLOS now publishes roughly 20,000 papers per year. TriLink researchers—including yours truly—are pleased to be PLOS authors in a November 2016 report titled Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation, which has been viewed more than 2,700 times as of June 2017.

Impossible Foods

Taken from ajitvadakayil.blogspot.com

Apparently pioneering Open Access wasn’t enough for Brown and in 2009 he decided to devote his sabbatical to a daunting—if not impossible—challenge: eliminating conventional meat production from animals, which he estimated to be the world’s largest environmental problem, according to a reported interview. Reducing meat consumption, Brown reasoned, would free up vast amounts of land and water, as well as mitigate climate change due to methane emitted by animals (specifically, 8% of the world’s water and 15% of greenhouse gas emissions, according to one report). In addition, there would be elimination of enormous quantities of chemical fertilizers that are harmful to water systems.

Taken from impossiblefoods

‘All you have to do is make a product that the current consumers of meat and dairy prefer to what they’re getting now,’ Brown said and succeeded in raising $3 million in venture capital seed money. His startup company—aptly named Impossible Foods—then raised $108 million, in a whopper of a deal (pun intended) for development of its initial product—a plant-based burger, also aptly named the Impossible Burger.

The Impossible Burger is made from all-natural ingredients such as wheat, coconut oil and potatoes. What makes this burger unlike all other veggie burgers is an ingredient called heme. Heme is commonly associated with hemoglobin—the red pigment in blood—but is also found in other hemoproteins, including those in plants, albeit in low abundance compared to red meat. Therein lies the part of this story I decided to research: how does Impossible Foods obtain large amounts of plant heme having the desired properties for their burger?

Leghemoglobin taken from web.mst.edu

In an Impossible Foods patent by Brown et al., I found soy leghemoglobin identified as one such exemplary heme, which is pictured right. Plant cells within the nodule produce leghemoglobin to serve as an oxygen carrier to the bacteria within the nodule, similar to hemoglobin in blood. This enables the bacteria to obtain enough oxygen for respiration but ensures that the oxygen is in a bound form so that it cannot harm nitrogen fixing enzymes inside the bacteria. Cutting open a nodule reveals the red color typical of leghemoglobin when it binds oxygen, as seen below.

Impossible Foods biomanufacturing facility. Taken from psmag.com

According to the patent, biosynthetic leghemoglobin was expressed and purified using recombinant DNA technology for protein production, and then shown by SDS-PAGE gel and mass spectrometry to be identical to soybean leghemoglobin isoforms purified from soybean root nodules. Given the advanced state-of-the-art of industrial-scale recombinant production, I assume the Impossible Foods processes pictured right can be scaled-up to reduce cost.

If you think that Brown’s burger probably falls short of what you’d want for a meat substitute, think again. After watching an unbiased—I assume—and rather entertaining video of numerous taste testers (including meat eaters and a life-long vegan) give it positive reviews, I set out to sample an Impossible Burger. It was served as two sliders topped with sun-dried tomatoes, cavolo nero, vegan sun-dried tomato mayonnaise on a poppy seed bun served with chickpea panelle. I found the taste and texture nicely meat-like, but the $16 cost a bit tough to swallow—pun intended.

Meat-Substitutes are Ethically Compelling and Becoming Big Business

Readers interested in the ethically compelling case for developing meat substitutes like the Impossible Burger may be interested in a newspaper story about a for-credit curriculum now offered by the University of California, Berkeley. In that article, I was particularly impressed by the extent of other investments in commercialization of meat-substitutes:

  • In direct competition with Impossible Foods, which has raised a total of $183 million, Beyond Meat (which counts both multibillionaire Bill Gates and meats giant Tyson Foods as investors), sells The Beyond Burger™ as well as other meatless products.
  • In 2014, Pinnacle Foods (Vlasic® pickles, Birds Eye® vegetables) bought meatless food producer Gardein for $154 million.
  • Last year, Monde Nissin (instant noodles, etc.) of the Philippines purchased US-based Quorn for $831 million. Quorn’s fungus-derived mycoprotein can be processed to look and taste like chicken nuggets, sausage or patties.
  • Some startups such as Mosa Meat in Holland and Perfect Day in Berkeley are pushing the genetic engineering toward completely biosynthetic “meat” and “milk,” respectively, as recently reported in The Economist.

In conclusion, I think you’ll agree with me that the aforementioned accomplishments of Pat O. Brown give him good reasons to be smiling so broadly in his picture above. I certainly would be.

As usual, your comments are welcomed.

Postscript

After writing this blog, I read about Memphis Meats in San Leandro, California, which—like Mosa Meat and Perfect Day—has been developing cell culture-based technology to produce “meat.” Focusing on chicken, the company is quoted as saying that ‘the taste and texture is similar to that of the real thing, just a bit spongier.’ While this seems promising, it currently costs around $9,000 to produce a pound of Memphis Meats’ poultry, compared to a bit over $3 for a pound of chicken breast. However, the company hopes to reduce costs drastically and to launch a commercial product in 2021. I hope it does, but I think it won’t.

SaveSave

SaveSaveSaveSave

SaveSave

SaveSave

SaveSave

SaveSave

National DNA Day 2016 – DNA Dreams Do Come True!

  • Khorana’s Dream of Synthesizing a Gene from Hand-Made Oligos
  • Caruther’s Dream of Automating Oligo Synthesis
  • Venter’s Dream of Fully Automating Gene Synthesis
  • Who’s Dreaming About What’s Next?

DNA Day ImageThis blog acknowledging National DNA Day on April 25th deals with dreams of various sorts, but mainly with gene synthesis, which was only a dream in the 1950s and is now achievable in a way few dreamed possible even a few years ago.

Before I get to DNA gene-dreams that did come true, I want to briefly mention two other dream-like anniversaries. First is the fact that my blog is now beginning its 4th year—yeh!—after its inaugural posting in April 2013 to celebrate 60 years since Watson & Crick’s famous publication of DNA’s helix structure as the fundamental basis for genetic material. Second is this year being TriLink’s 20th anniversary—yeh!—as a leading provider of modified nucleic acids, which co-founders Rick Hogrefe and Terry Beck likely view as their business dream come true. But I digress…

The First Dreamer and Doer Continue reading

Science vs. Semantics—Perspectives Matter When it Comes to Genetically Modified and Genetically Edited Organisms

  • GMO Science and Regulation Have Created Genetic Gordian Knot
  • New Genetic Editing Methods Outpace Rules for Consumer Protection
  • Marker-Based Breeding May Bypass GMOs
  • Soylent Green No Longer A Futuristic Concept
The legendary Gordian knot is a metaphor for an intractable problem. Taken from counter-current.com.

The legendary Gordian knot is a metaphor for an intractable problem. Taken from counter-current.com.

Truth be told, I’ve been vacillating for a long time about writing this blog about genetically modified organisms (GMOs), not for lack of relevance to what’s trending in nucleic acids research, but because it’s an exceedingly complicated subject with no definitive conclusions. There’s a complex mixture of underlying genetic methods, overriding regulatory issues, confused-consumer viewpoints, and balancing global ecosystem vs. humanitarian needs—all interwoven into a modern day version of the Gordian knot.

I’ll now try to unravel some of these tangled perspectives largely to comment on newish nucleic acid-based techniques that are forcing a rethink of regulations in order to better deal with what has become a “regional rat’s nest of regulatory gobbledygook.” Intermixed with the gobbledygook are well-intentioned advocacy groups that nevertheless seem guilty of using irrational—to me—consumer scare tactics.

Continue reading

De-Extinction: Hope or Hype?

  • Can scientists “revive” woolly mammoths?
  • Passenger Pigeons, possibly?
  • Is “facilitated adaption” more realistic?

If you haven’t seen the 1993 movie Jurassic Park, the plot involves a tropical island theme park populated with cloned dinosaurs created by a bioengineering company, InGen. The cloning was accomplished by extracting the DNA of dinosaurs from mosquitoes that had been preserved in amber—not unlike extraction of ancient yeast DNA from extinct bees preserved in amber for brewing “Jurassic beer” that I featured in a previous posting. However, in Jurassic Park the strands of DNA were incomplete, so DNA from frogs was used to fill in the gaps. The dinosaurs were cloned genetically as females in order to prevent breeding.

This is all a great premise for a movie, but will Jurassic Park-like fantasy become reality in the near future?  What’s being investigated now, and are there concerns being voiced? These are just some of the questions touch upon below.

Woolly Mammoths May One Day Roam Real-Life Jurassic Park

Hendrik Poinar, Director of the Ancient DNA Centre at McMaster University in Hamilton, Ontario (taken from fhs.mcmaster.ca via Bing Images).

Dr. Hendrik Poinar, Director of the Ancient DNA Centre at McMaster University (taken from fhs.mcmaster.ca).

Dr. Hendrik Poinar, Associate Professor at McMaster University in Canada, was trained as a molecular evolutionary geneticist and biological anthropologist, and now specializes in novel techniques to extract and analyze “molecular information (DNA and/or protein sequences)” from ancient samples. His work included such projects as sequencing the mitochondrial genome of woolly mammoths that went extinct long ago. Based on that work, Dr. Poinar was recently interviewed by CBC News about the likelihood of reestablishing woolly mammoths. Here are some excerpts:

Q: Without getting too technical, describe what you’re doing to bring back animals like the woolly mammoth?

A: We’re interested in the evolutionary history of these beasts. These lumbering animals lived about 10,000 years ago and went extinct. We’ve been recreating their genome in order to understand their origins and migrations and their extinction. That led to the inevitable discussion about if we could revive an extinct species and is it a good thing.

Q: Why is this so interesting to you?

A: There are reasons why these animals went extinct. It could be climate, it could be human-induced over-hunting. If we can understand the processes that caused extinction, maybe we can avoid them for current endangered species. Maybe we need to think about what we can do to bring back extinct species and restore ecosystems that are now dwindling.

Q: Is it possible to bring these things back to life?

A: Not now. We’re looking at 30 to 50 years.

Woolly mammoths roamed both North America and Asia for hundreds of thousands of years. Many went extinct during the most recent period of global warming (taken from CBC News via Bing Images).

Woolly mammoths roamed both North America and Asia for hundreds of thousands of years. Many went extinct during the most recent period of global warming (taken from CBC News via Bing Images).

Q: How would you do something like that?

A: First thing you have to do is to get the entire blueprint. We have mapped the genome of the woolly mammoth. We’re almost completely done with that as well as a couple other extinct animals. We can look at the discrete differences between a mammoth and an Asian elephant. We would take an Asian elephant chromosome and modify it with mammoth information. Technology at Harvard can actually do that. Take the modified chromosomes and put them into an Asian elephant egg. Inseminate that egg and put that into an Asian elephant and take it to term. It could be as soon as 20 years.

Q: Is this such a good idea?

A: That’s the million-dollar question. We’re not talking about dinosaurs. We’ll start with the herbivores—the non-meat eaters. We could use the technology to re-introduce diversity to populations that are dwindling like the cheetah or a wolf species we know are on the verge of extinction. Could we make them less susceptible to disease? Is it good for the environment? We know that the mammoths were disproportionately important to ecosystems. All the plant species survived on the backs of these animals. If we brought the mammoth back to Siberia, maybe that would be good for the ecosystems that are changing because of climate change.

Q: You are tinkering with the evolutionary process?

A: Yes, but would you feel differently if the extinction was caused by man like it was with the passenger pigeon or the Tasmanian wolf, which were killed by humans? Even the large mammoth, there are two theories on their extinction, one is overhunting by humans…and the other is climate. Do we have a moral obligation?

Bringing Back Passenger Pigeons

Ben Novak has a BS in Ecology and worked with mastodon fossils toward a master’s degree at McMaster University, but he abandoned that to pursue his long-time passion for passenger-pigeon genetics (taken from wfs.org via Bing Images).

Ben Novak has a BS in Ecology and worked with mastodon fossils toward a master’s degree at McMaster University, but he abandoned that to pursue his long-time passion for passenger-pigeon genetics (taken from wfs.org via Bing Images).

Ben Novak, according to an interview in Nature last year, has spent his young career endeavoring to resurrect extinct species. Although he has no graduate degree, he has amassed the skills and funding to start a project to bring back the Passenger Pigeon—once the United States’ most numerous bird (about 5 billion according to Audubon)—which died out in 1914. Following are comments from Ben, taken from the Nature article referenced above, about how his work is funded and its prospects.

“Once I had passenger-pigeon tissue [from the Field Museum of natural History in Chicago, Illinois], I started applying for grants to do population analysis, but I couldn’t secure funding. I got about $4,000 from family and friends to sequence the DNA of the samples. When I got data, I contacted George Church, a molecular geneticist at Harvard Medical School in Boston, Massachusetts, who was working in this area. He and members of Long Now Foundation in San Francisco, California, which fosters long-term thinking, were planning a meeting on reviving the passenger pigeon….The more we talked, the more they discovered how passionate I was. Eventually, Long Now offered me full-time work so that nothing was standing in my way.”

“I have just moved to the University of California, Santa Cruz, to work with Beth Shapiro. She has her own sample of passenger pigeons, and we want to do population genetics and the genome. It’s a good fit. Long Now pays me, and we do the work in her lab, taking advantage of her team’s expertise in genome assemblies and ancient DNA.”

Male passenger pigeon (taken from swiftbirder.wordpress.com via Bing Images).

Male passenger pigeon (taken from swiftbirder.wordpress.com via Bing Images).

For the sad story of how this creature went extinct, click here to access an account written by Edward Howe Forbush in 1917.

Doing more searching about Ben Novak led me to another 2013 interview, this time in Audubon. When asked if it’s realistic to get a healthy population from a few museum specimens, here’s what he said.

“If we’re willing to create one individual [passenger pigeon], then through the same process we can produce individuals belonging to completely different genetic families. We can make 10 individuals that, when they’re mated, will have an inbreeding coefficient near zero…First we need to discern what the actual genetic structure of the species was. We can analyze enough tissue samples to get that genetic diversity.”

While perusing the Long Now Foundation’s website, I was pleased to read a Passenger-Pigeon progress report posted by Ben Novak on October 18th 2013.  The posting gives a detailed update on genomic sequencing of “Passenger Pigeon 1871″ [date of preservation] at the University of California San Francisco‘s Mission Bay campus sequencing facility, as well as some nice pictures. Given what he said above about 10 individuals being theoretically adequate for reviving and restoring an extinct population, you’ll be as pleased as Ben is about the following.

“Passenger Pigeon 1871 was selected as the candidate for the full genome sequence for its superb quality compared to other passenger pigeon specimens. Over the last two years Dr. Shapiro, myself and colleagues have scrutinized the quality of 77 specimens including bones and tissues. Our first glimpses of data confirmed that the samples would be able to provide the DNA needed for a full genome sequence, but as we delved into the work, the specimens exceeded our expectations. Not only do we have one specimen of high enough quality for a full genome, we have more than 20 specimens to perform population biology research with bits of DNA from all over the genome.”

Revive and Restore

Reading about Ben Novak’s support from the Long Now Foundation led me discover the organization’s Revive and Restore Project, aimed at genetic rescue of endangered and extinct species. Its mission is stated as follows:

“Thanks to the rapid advance of genomic technology, new tools are emerging for conservation. Endangered species that have lost their crucial genetic diversity may be restored to reproductive health. Those threatened by invasive diseases may be able to acquire genetic disease-resistance.

It may even be possible to bring some extinct species back to life. The DNA of many extinct creatures is well preserved in museum specimens and some fossils. Their full genomes can now be read and analyzed. That data may be transferable as working genes into their closest living relatives, effectively bringing the extinct species back to life. The ultimate aim is to restore them to their former home in the wild.

Molecular biologists and conservation biologists all over the world are working on these techniques. The role of Revive and Restore is to help coordinate their efforts so that genomic conservation can move ahead with the best current science, plenty of public transparency, and the overall goal of enhancing biodiversity and ecological health worldwide.”

This Project’s website is well worth visiting, as it provides a fascinating mix of species under consideration (such as the Passenger Pigeon and the woolly mammoth), various video presentations by advocates, and an engaging blog. It also provides a very convenient “donate” button should you be so inclined.

While the Passenger Pigeon project and other Revive and Restore efforts are well intended, I’m more inclined at this time to be neutral-to-negative about the projects, and will reserve a final opinion until all parties, pro and con, have extensive debates similar to what was done in the past for then (and still) controversial recombinant DNA technology. Given the amount of concern and caution then for what we can now view as conventional genetic engineering, it seems reasonable to me that, with far more powerful tools for genomics and synthetic biology being available, “an abundance of caution” is in order when dealing with the possibility of resurrecting extinct species. If Jurassic Park serves as any sort of model for what science can accomplish, perhaps we should also consider what the movie highlights as the potential implications of those accomplishments.

For now, I’m intently interested in the continuing debates and I find it fascinating to consider alternatives such as rescuing species from extinction as outlined next.

“Facilitated Adaption” Pros & Cons

Michael A. Thomas, Professor of Biology at Idaho State University, and colleagues authored a Comment in Nature last year entitled Gene tweaking for conservation that is freely available (yeh!) and well worth reading. Some highlights are as follows:

Sadly, if not shockingly, conservative estimates predict that 15–40% of living species will be effectively extinct by 2050 as a result of climate change, habitat loss and other consequences of human activities. Among the interventions being debated, facilitated adaptation has been little discussed. It would involve rescuing a target population or species by endowing it with adaptive alleles, or gene variants, using genetic engineering—not too unlike genetically modified crops that now occupy 12% of today’s arable land worldwide. Three options for facilitated adaption are outlined.

“Poster Child” for facilitated adaption: an endangered Florida panther population was bolstered through hybridization with a related subspecies — a technique that could be refined using genomic tools (taken from Thomas et al. Nature 2013).

“Poster Child” for facilitated adaption: an endangered Florida panther population was bolstered through hybridization with a related subspecies — a technique that could be refined using genomic tools (taken from Thomas et al. Nature 2013).

First, threatened populations could be cross with individuals of the same species from better-adapted populations to introduce beneficial alleles. A good example of this is crossing a remnant Florida panther population with related subspecies from Texas that significantly boosted the former population and its heterozygosity, a measure of genetic variation that was desired. Risks of this approach include dilution of locally adaptive alleles.

Second, specific alleles taken from a well-adapted population could be spliced into the genomes of threatened populations of the same species. This was exemplified by recent work wherein heat-tolerance alleles in a commercial trout were identified for possible insertion into fish eggs in populations threatened by rising water temperature. Such an approach was viewed as low risk because it involves genetic manipulations within the same species.

Third, genes removed from a well-adapted species could be incorporated into the genomes of endangered individuals of a different species. This transgenic approach has been extensively used to improve plant crops toward drought and temperature. However, outcomes are hard to predict, and a major concern is that such an approach could bring unintended and unmanageable consequences—definitely a scary possibility.

What do you think about reintroducing extinct species?  Do you see other pros and cons to facilitated adaption?  As always, your comments are welcomed.

Postscript

The following, entitled ‘De-Evolving’ Dinosaurs from Birds, recently appeared in GenomeWeb:

Ancient animals could be resurrected through the genomes of their modern-day descendants, Alison Woollard, an Oxford biochemist tells the UK’s Daily Telegraph. For instance, the DNA of birds could be “de-evolved” to resemble the DNA of dinosaurs, the paper adds.

“We know that birds are the direct descendants of dinosaurs, as proven by an unbroken line of fossils which tracks the evolution of the lineage from creatures such as the velociraptor or T-Rex through to the birds flying around today,” Woollard says, later adding that “[i]n theory we could use our knowledge of the genetic relationship of birds to dinosaurs to ‘design’ the genome of a dinosaur.”

In both the book and movie Jurassic Park, the fictional resurrection of dinosaurs relied on dinosaur DNA that was preserved in fossilized biting insects, but as the Daily Telegraph notes, a study in PLOS One earlier this year found no evidence of DNA from amber-preserved insects.

Daily Telegraph adds that any dinosaur DNA recovered from bird genomes would be fragmented and difficult to piece back together. A mammoth, it says, might have a better shot.

The Buzz on the Cut: From Dream to Reality

Targeted Genome Engineering with Zinc-finger Nucleases, TALENs and CRISPR

Targeted genome editing tools such as meganucleases, zinc-finger nucleases, TALENs and CRISPR are among the hottest topics in cell and gene therapy. Dr. Anton McCaffrey, Principal Scientist at TriLink and expert in these areas, gives herein his overview after attending the recent American Society for Gene and Cell Therapy Meeting (May 2013) and the International Society for Stem Cell Research Meeting (June 2013) where there were a number of exciting talks discussing applications of this technology.

amccaffrey


Dr. McCaffrey received his PhD in Biochemistry from the University of Colorado at Boulder in 1999. During his postdoctoral fellowship at Stanford he developed gene therapeutics for hepatitis B and C. He was then Assistant Professor at
University of Iowa where he focused on the role of microRNAs during the pathogenesis of hepatitis C virus and developed RNA interference and zinc-finger nuclease based therapeutics for treatment of hepatitis B virus. Now he manages the RNA Transcription product line at TriLink.

So what is targeted genome engineering with nucleases and why would you want to do this?  The primary goal in cells or animals is to create a specific, localized double-stranded DNA break and then to: 1. correct the sequence of a defective targeted gene, 2. knock in a specific gene mutation to create a disease model or 3. knock out a gene. The basic idea is to rationally design artificial restriction enzymes that recognize a specific location within the DNA genome of a cell or organism and catalyze a double stranded break at this location (Figure 1).

In the first two cases, where the target gene is to be specifically edited, the nuclease(s) are co-transfected with an exogenous donor DNA molecule. This donor DNA contains arms, which share homology with the target loci and will direct homologous recombination at the targeted cut site. The sequence of the donor DNA replaces that of the endogenous locus at one or both alleles. So, for example, a wild-type donor sequence can be used to replace a mutated gene sequence to correct a genetic disease.

If one wishes to inactivate a gene using these technologies, an exogenous donor template is not included. In the absence of a donor, the cell uses non-homologous end joining to repair the double stranded break. At a high frequency, this process introduces deletions and insertions in the gene, which changes the reading frame and inactivates the gene.

Until the advent of these technologies, it was impossible to make transgenic animals other than mice. Using targeting genome engineering is now possible to make transgenic rats, pigs, ferrets and plants. As will be discussed below, advances in messenger RNA (mRNA)-based gene therapy are converging with advances in targeted genome engineering to enable efficient, yet transient expression of designer nucleases without risk of undesired integration of the nuclease expression vector.

Figure 1.  Nuclease Mediated Double Stranded Breaks Stimulate Homologous Gene Replacement or Targeted Gene Inactivation.  If targeted nucleases are co-transfected with a homologous donor DNA fragment, homologous recombination replaces defective DNA with a corrected sequence (left).  In the absence of a DNA donor fragment, non-homologous end joining repairs the break, but with frequent insertions and deletions, thus inactivating the gene.


Figure 1. Nuclease Mediated Double Stranded Breaks Stimulate Homologous Gene Replacement or Targeted Gene Inactivation. If targeted nucleases are co-transfected with a homologous donor DNA fragment, homologous recombination replaces defective DNA with a corrected sequence (left). In the absence of a DNA donor fragment, non-homologous end joining repairs the break, but with frequent insertions and deletions, thus inactivating the gene.

Meganucleases

Techniques for making targeted nucleases are rapidly evolving.  Initial attempts to engineer designer restriction nucleases to target new sequences revolved around changing the specificity of naturally occurring nucleases such as meganucleases.  Meganucleases are restriction enzymes with long recognition sites (12-40 nucleotides). These nucleases could be engineered to recognize related sequences in genomes and cleave them.  However, only a small number of sites could be targeted using this approach (refs A-C).

Zinc-finger nucleases (ZFNs)

Zinc-finger nucleases (ZFNs) were the next major advance in the field. Zinc fingers are the most common DNA binding motif in mammalian transcription factors. These sequence specific binding domains can be engineered to bind to novel DNA sequences. Zinc-fingers can be turned into nucleases by fusing them to non-specific cleavage domains, such as the FokI nuclease. FokI cleaves as a dimer, so pairs of ZFNs are designed to bind to adjacent sites in the genome to allow FokI dimer formation and double stranded DNA cleavage (Figure 2). A number of laboratories published design rules that serve as a starting point to engineer ZFNs with novel DNA binding specificities (refs N-R). In reality, actual binding specificity is context dependent. Several selection protocols in cells also exist for identifying novel ZFNs. ZFNs have been successfully used to modify the genomes of Drosophila, C. elegans, zebrafish and rats (refs D-M). However, identification of functional ZFNs remains challenging and most ZFNs have emerged from a small number of laboratories with specialist skills.

 Figure 2. Zinc-Finger Nucleases Bind as Dimers to Cut Double Stranded DNA. Adapted from Gaj et al.Trends Biotechnol. 2013 May 8. [Epub ahead of print]


Figure 2. Zinc-Finger Nucleases Bind as Dimers to Cut Double Stranded DNA. Adapted from Gaj et al.Trends Biotechnol. 2013 May 8. [Epub ahead of print]

Transcription activator-like effector nuclease (TALENs)

In the last few years, TALENs have emerged as a more generally accessible alternative to ZFNs. Like ZFNs, TALENs utilize a modular DNA binding motif (TALE) that can be modified to introduce new DNA binding specificities. TALENs consist of multiple repeat variable diresidues (RVDs) which each specify binding to a single nucleotide (Refs S-U).  TALEN arrays are made by stringing together RVDs in a specific order to provide specificity and binding affinity to novel DNA sequences. Commonly, engineered TALE sequences are fused to non-specific cleavage domains such as FokI. As with ZFNs, TALENs function as pairs bound to adjacent DNA sequences. Unlike ZFNs, TALENs are not as prone to sequence context effects, which greatly complicate the de novo design of ZFNs. This has made them much more accessible to the general scientific community. A number of groups have published TALEN assembly protocols that allow assembly of these repetitive sequences, including one popular open source assembly method is known as Golden Gate (Refs V-Y).

 Figure 3. TALENs Bind as Dimers to Cut Double Stranded DNA. Adapted from Gaj et al.Trends Biotechnol. 2013 May 8. [Epub ahead of print]


Figure 3. TALENs Bind as Dimers to Cut Double Stranded DNA. Adapted from Gaj et al.Trends Biotechnol. 2013 May 8. [Epub ahead of print]

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

The newest kid on the genome-engineering block is CRISPR. CRISPR is a bacterial immune system in which bacteria sample the DNA of pathogens, integrate foreign DNA into their genome in specialized repeat structures, and then use these sequences to produce Guide RNAs that direct cutting of homologous pathogenic DNA sequences. To some degree this is reminiscent of RNA interference in mammals. Once the target site has been delineated by the RNA guide sequence, Cas proteins (CRISPR-associated proteins) do the cutting. A number of groups have adapted this system to create RNA directed genome engineering tools (refs Z-CC) (Figure 4). This new system has generated considerable interest since recognition of the target DNA sequence to be cut is RNA mediated rather than protein mediated. DNA cleavage is carried out by the expressed Cas9 protein. With ZFNs and TALENs, if you wish to target a new site, you have to identify and synthesize two new proteins. With CRISPR, you use the same Cas9 protein each time and just alter the sequence of the guide RNA. Stay tuned for head to head comparisons of the efficiency and specificity of ZFNs, TALENs and CRISPR that are about to be published.

 Figure 4. CRISPR is an RNA Guided Genome Engineering System.  Figure adapted from DiCarlo et al. Nucleic Acids Research, 2013, Vol. 41, No. 7.


Figure 4. CRISPR is an RNA Guided Genome Engineering System. Figure adapted from DiCarlo et al. Nucleic Acids Research, 2013, Vol. 41, No. 7.

Modified mRNA for Transient Expression in Genome Engineering

In each of the three systems described above, one needs to express one or two proteins inside cells or an organism.  Plasmids and viral vectors have been used to achieve this, but these carry a risk. Double stranded DNA breaks catalyze insertion of DNA at the cut site.  At some substantial frequency, the protein expression vectors can integrate at the cut site. These vectors necessarily carry eukaryotic promoters, which can lead to continuous expression of the nuclease or the expression of previously silent sequences. For clinical applications this can be a major issue. One also needs to consider off-target cleavage of by engineered nucleases. Since ZFNs have been around longer than TALENs or CRISPR, the most data exists for ZFNs. It is clear that ZFNs can cut at pseudo-sites that resemble the chosen target site.For this reason, transient expression of nucleases is desirable. Many in the ZFN and TALEN field have moved to expression of these nucleases from synthetic mRNAs because they are transient and have no risk of insertion.  Synthetic mRNAs, which mimic fully processed, capped and polyadenylated mRNAs, can be produced in large quantities by in vitro transcription. Transfected mRNAs made with adenine, cytosine, guanine and uracil are recognized as pathogens by innate immune sensors such as Toll-like receptors, RIG-I and PKR. Kariko et al. showed that mRNAs could be made much less immunogenic and non-toxic by substitution of cytosine and uridine with 5-methylcytosine and pseudouridine (ref DD). Custom syntheses of milligram to gram amounts of 5-methylcytosine and pseudouridine modified mRNAs can be ordered from TriLink BioTechnologies. Cas9 mRNA is also available as a catalog item.

Conclusions

In recent years, designer genome engineering has gone from dream to reality. New editing systems are taking this from the realm of a few elite laboratories and companies to democratizing it for the masses. Concurrent advances in mRNA gene therapy are providing safe and effective delivery systems for expressing the necessary components in cells and animals. There is now huge interest in using targeted genome engineering in patient derived somatic cells and stem cells. Rather than simply knocking genes in or knocking them out, we may now be able to actually correct monogenic genetic disorders.  Clinical trials are currently under way to determine if ZFNs can be used to inactivate the CCR5 HIV co-receptor to make patient T-cells immune to HIV. These technologies will also enable facile creation of disease models in species other than mice. The future is bright for targeted genome engineering. That’s the buzz on the cut.

A sincere thanks to Anton McCaffrey for providing this update on truly exciting trends in nucleic acid-based technologie. As always, I welcome comments and discussions.

References

A. I-SceI-induced gene replacement at a natural locus in embryonic stem cells. Cohen-Tannoudji M, Robine S, Choulika A, et al. Mol Cell Biol 1998;18:1444-8.

B. The yeast I-Sce I meganuclease induces site-directed chromosomal recombination in mammalian cells. Choulika A, Perrin A, Dujon B, Nicolas JF. C R Acad Sci III 1994;317:1013-9.

C. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Choulika A, Perrin A, Dujon B, Nicolas JF. Mol Cell Biol 1995;15:1968-73.

ZFNs modifying different organisms

D. Efficient gene targeting in Drosophila with zinc-finger nucleases. Beumer K, Bhattacharyya G, Bibikova M, Trautman JK, Carroll D. Genetics 2006;172:2391-403.

E. Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Beumer KJ, Trautman JK, Bozas A, et al. Proc Natl Acad Sci U S A 2008;105:19821-6.

F. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Bibikova M, Golic M, Golic KG, Carroll D. Genetics 2002;161:1169-75.

G. Genetic Analysis of Zinc-finger Nuclease-induced Gene Targeting in Drosophila. Bozas A, Beumer KJ, Trautman JK, Carroll D. Genetics 2009.

H. Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells. Morton J, Davis MW, Jorgensen EM, Carroll D. Proc Natl Acad Sci U S A 2006;103:16370-5.

I. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Doyon Y, McCammon JM, Miller JC, et al. Nat Biotechnol 2008;26:702-8.

J. Zinc finger-based knockout punches for zebrafish genes. Ekker SC. Zebrafish 2008;5:121-3.

K. Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN). Foley JE, Yeh JR, Maeder ML, et al. PLoS ONE 2009;4:e4348.

L. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Meng X, Noyes MB, Zhu LJ, Lawson ND, Wolfe SA. Nat Biotechnol 2008;26:695-701.

M. Knockout rats via embryo microinjection of zinc-finger nucleases. Geurts AM, Cost GJ, Freyvert Y, et al. Science 2009;325:433.

ZFN design rules

N. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences. Segal DJ, Dreier B, Beerli RR, Barbas CF, 3rd. Proc Natl Acad Sci U S A 1999;96:2758-63.

O. Insights into the molecular recognition of the 5′-GNN-3′ family of DNA sequences by zinc finger domains. Dreier B, Segal DJ, Barbas CF, 3rd. J Mol Biol 2000;303:489-502.

P. Validated zinc finger protein designs for all 16 GNN DNA triplet targets. Liu Q, Xia Z, Zhong X, Case CC. J Biol Chem 2002;277:3850-6.

Q. Development of zinc finger domains for recognition of the 5′-ANN-3′ family of DNA sequences and their use in the construction of artificial transcription factors. Dreier B, Beerli RR, Segal DJ, Flippin JD, Barbas CF, 3rd. J Biol Chem 2001;276:29466-78.

R. Development of zinc finger domains for recognition of the 5′-CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. Dreier B, Fuller RP, Segal DJ, et al. J Biol Chem 2005;280:35588-97.

TALENS

Breaking the code of DNA binding specificity of TAL-type III effectors. S. Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A. and Bonas, U. Science 2009;326,1509-12.

T.  The crystal structure of TAL effector PthXo1 bound to its DNA target. Mak, A.N., Bradley, P., Cernadas, R.A., Bogdanove, A.J. and Stoddard, B.L. Science 2012;335, 716-9.

U. A simple cipher governs DNA recognition by TAL effectors. Moscou, M.J. and Bogdanove, A.J. Science 2009;326,1501.

Golden gate

V. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A., Somia, N.V., Bogdanove, A.J., Voytas, D.F., Geissler et al. Nucleic Acids Res 2011;39,e82.

W. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Li, T., Huang, S., Zhao, X., Wright, D.A., Carpenter, S., Spalding, M.H., Weeks, D.P. and Yang, B., Morbitzer et al. Nucleic Acids Res 2011;39, 6315-25.

X. A modular cloning system for standardized assembly of multigene constructs. Weber, E., Engler, C., Gruetzner, R., Werner, S. and Marillonnet, S. PLoS One 2011;6, e16765.

Y. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G.M. and Arlotta, P. Nat Biotechnol 2011;29,149-53.

CRISPR

Z. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Jinek, M; Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. Science 2012;PMID 22745249.

AA. Multiplex genome engineering using CRISPR/Cas systems. Cong, Le; Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Science 2013;PMID 23287718.

BB. RNA-guided human genome engineering via Cas9. Mali, P; Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. Science 2013;PMID 23287722.

CC. One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Wang, H; Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. Cell 2013;PMID 23643243.

Modified mRNA

DD. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D. Mol Ther. 2008;Nov;16(11):1833-40.