It’s not every day that something from the 17th century gets radically reinvented.
But this month, a team from the Broad Institute at MIT and Harvard took aim at one of the most iconic pieces of lab ware—the microscope—and tore down the entire concept to recreate it from scratch.
I’m sure you have a mental picture of a scope: a stage to put samples on, a bunch of dials to focus the image, tunnel-like objectives with optical bits, an eyepiece to observe the final blown-up image. Got it? Now erase all that from your mind.
The new technique, dubbed DNA microscopy, uses only a pipette and some liquid reagents. Rather than monitoring photons, here the team relies on “bar codes” that chemically tag onto biomolecules. Like cell phone towers, the tags amplify, broadcasting their signals outward. An algorithm can then piece together the captured location data and transform those GPS-like digits into rainbow-colored photos.
The results are absolutely breathtaking. Cells shine like stars in a nebula, each pseudo-colored according to their genomic profiles.
That’s the crux: DNA microscopy isn’t here to replace its classic optical big brother. Rather, it pulls the viewer down to a molecular scale, allowing you to see things from the “eyes of the cell,” said study author Dr. Joshua Weinstein, who worked under the supervision of Dr. Aviv Regev and Dr. Feng Zhang, both Howard Hughes Medical Institute investigators.
The tech decodes the natural location of molecules inside a cell—and how they interact—while simultaneously gathering information about its gene expression in a two-in-one combo. It’s a bonanza for scientists struggling to tease apart individual differences in cells that physically look identical—say, immune cells that secrete different antibodies, or cancer cells in their early stage of malignant transformation.
“It’s a completely new way of visualizing biology,” said Weinstein in a press release. “This gives us another layer of biology that we haven’t been able to see.”
Almost all current microscopy techniques stem from the original all-in-one light microscope, first introduced in the 17th century. The core concept is light: the device guides photons refracted from the sample into a series of mirrors and optical lenses, resulting in an enlarged image of whatever you’re focusing on. It basically works like a DSLR camera with a very powerful zoom lens.
Scientists have long since moved past the visible light spectrum. Electron microscopy, for example, measures electrons that bounce off tissue to look at components inside the cell. Fluorescent microscopy—the “crown prince” of imaging—captures emitted light waves after stimulating tissue-bound fluorescent probes with lasers.
But here’s the thing: even as traditional microscopy is increasingly perfected and streamlined, it hits two hard limits. One is resolution. Light scatters, and there’s only so much we can do to focus the beam on one point to generate a clear image. This is why a light microscope can’t clearly see DNA molecules or proteins—it’s like using a smartphone to capture a single bright star. As a result, current microscopes generate satellite views of goings-on on the cellular “Earth.” Sharp, but from afar.
The second is genomic profiling. There’s been a revolution in mapping cellular diversity to uncover how visually similar cells can harbor dramatically different genomic profiles. A perfect example is the immune system. Immune cells that look similar can secrete vastly different antibodies, or generate different protein “arms” on their surface to grab onto specific types of cancer cells. Sequencing the cells’ DNA loses spatial information, making it hard to tease out whether location is important for designing treatments.
So far, microscopy has only focused on half of the picture. With DNA microscopy, the team hopes to grab the entire landscape.
“It will allow us to see how genetically unique cells—those comprising the immune system, cancer, or the gut, for instance—interact with one another and give rise to complex multicellular life,” explained Weinstein.
To build their chemical microscope, the team began with a group of cultured cells.
They decoded the cells’ RNA molecules and transcribed the data to generate a complete library of expressed genes called cDNA. Based on cDNA sequences, they then synthesized a handful of tags randomly made of the DNA letters A, T, C, and G, each about 30 letters long. When bathed onto a new batch of cells, the tags tunneled inside and latched onto specific RNA molecules, labeling each with a specific barcode.
To amplify individual signals—each “point source”—the team used a common chemical reaction that rapidly amplifies DNA molecules, increasing their local concentration. DNA doesn’t like staying put inside the liquid interior of a cell, so the tags slowly begin to drift outwards, like a drop of dye expanding in a pool of water. Eventually, the DNA tags balloon into a molecular cloud that stems from their initial source on the biomolecule.
“Think of it as a radio tower broadcasting its signal,” the authors explained.
As DNA tag clouds from multiple points grow, eventually they’ll collide. This triggers a second reaction: two diffusing DNA molecules physically link up, spawning a unique DNA label that journals their date. This clever hack allows researchers to eventually triangulate the location of the initial sources: the closer the two points are in the beginning, the more labels they’ll form. The further apart, the fewer. The idea is very similar to cell phone companies using radio towers to track their users’ locations, in which they measure where signals from three or more towers intersect.
The cells are subsequently collected and their DNA extracted and sequenced—something that takes roughly 30 hours. The data is then decoded using a custom algorithm to transform raw data into gorgeous images. This is seriously nuts: the algorithm has absolutely no clue what a “cell” is, but still identified individual cells at their location inside the sample.
“The first time I saw a DNA microscopy image, it blew me away,” said Regev.
As proof of concept, the team used the technique to track cells that encode either green or red fluorescent proteins. Without any previous knowledge of their distribution, the DNA microscope efficiently parsed the two cell types, although the final images were blurrier than that obtained with a light microscope. The tech could also reliably map the location of individual human cancer cells by tagging the cells’ internal RNA molecules, although it couldn’t parse out fine details inside the cell.
A Whole New World
Thanks to DNA’s “stickiness,” the technique can be used to label multiple types of biomolecules, allowing researchers to track the location of and identify antibodies or surface proteins on any given cell type, although the team will have to further prove its effectiveness in tissue samples.
Although the resolution of DNA microscopy is currently on par, if not slightly lower than, that of a light microscope, it provides an entirely new perspective of biomolecules inside cells.
“We’ve used DNA in a way that’s mathematically similar to photons in light microscopy. This allows us to visualize biology as cells see it and not as the human eye does,” said Weinstein.
DNA microscopy already does things a light microscope can’t. It can parse out cells that visually look similar but have different genetic profiles, for example, which comes in handy for identifying various types of cancer and immune cells. Another example is neuroscience: as our brains develop, various cells drastically alter their genetic profiles while migrating long distances—DNA microscopy could allow researchers to precisely track their movements, potentially uncovering new ways to boost neuroregeneration or plasticity.
Only time will tell if DNA microscopy will reveal “previously inaccessible layers of information,” as the team hopes. But they believe that their invention will spark new ideas and uses in the scientific community.
“It’s not just a new technique, it’s a way of doing things that we haven’t ever considered doing before,” said Regev.
An army of free-floating minibrain clones are heading your way!
No, that’s not the premise of a classic sci-fi brain-in-jars blockbuster. Rather, a team at Harvard has figured out a way to “clone” brain organoids, in the sense that every brain blob, regardless of what type of stem cell it originated from, developed nearly identically into tissues that mimic the fetal human cortex. No, they didn’t copy-paste one minibrain to make a bunch more—rather, the team found a recipe to reliably cook up hundreds of 3D brain models with very little variability in their cellular constituents.
If that sounds like a big ole “meh,” think of it like this. Minibrains, much like the organs they imitate, are delicate snowflakes, each with their own unique cellular makeup. Sure, no two human brains are exactly the same, even those of twins. However, our noggins do follow a relatively set pathway in initial development and end up with predictable structures, cell types, and connections.
Not so for minibrains. “Until now, each … makes its own special mix of cell types in a way that could not have been predicted at the outset,” explained study author Dr. Paola Arlotta. By compiling a cellular atlas from multiple minibrains, her team basically found a blueprint that coaxes stem cells from different genetic and gender origins to mature into remarkably similar structures, at least in terms of cellular composition. Putting it another way, they farmed a bunch of identical siblings; but rather than people, they’re free-floating brain blobs.
Rest assured, it’s not a new evil-scientist-brain-control scheme. For brain organoids to be useful in neuroscience—for example, understanding how autism or schizophrenia emerge—scientists need large amounts of near-identical test subjects. This is why twins are extremely valuable in research: all else (nearly) equal, they help isolate the effects of individual treatments or environmental changes.
“It is now possible to compare ‘control’ organoids with ones we create with mutations we know to be associated with the disease,” said Arlotta. “This will give us a lot more certainty about which differences are meaningful.”
How to Grow a Brain
The authors set out with a slightly different question in mind: is it possible to reliably grow a brain outside the womb?
You may be asking “why not?” After all, scientists have been cooking up brain organoids for half a decade. But although specific instructions are generally similar, the resulting minibrains—not so much.
Here’s the classic recipe. Scientists start with harvested stem cells, embed them into gel-like scaffolds, then carefully nurture them in a chemical soup tailored to push the cells to divide, migrate, and mature into tiny balls. These tissue nuggets are then transferred to a slow spinning bioreactor—imagine a giant high-tech smoothie machine. The gentle whirling motion keeps the liquid nicely oxygenated. In six months, the grains of greyish tissue expand to a few millimeters, or one-tenth of the width of your finger, packed full with interconnected brain cells.
This is the “throw all ingredients into a pot and see what happens” approach, more academically known as the unguided method. Because scientists don’t interfere with the brain balls’ growth, the protocol gives stem cells the most freedom to self-organize. It also allows stem cells to stochastically choose what type of brain cell—neurons, glia, immune cells—they eventually become. God may not play dice, but outside the womb, stem cells sure do.
That’s problematic. Depending on the initial stem cell population, the culture conditions, and even the particular batch, the resulting minibrains end up with highly unpredictable proportions of cell types arranged in unique ways. This makes controlled experimenting with minibrains extremely difficult, which nixes their whole raison d’être.
Similar to their human counterparts, unguided minibrains also follow the instructions laid out in their DNA. So what gives?
Our brains do not grow in isolation. Rather, they’re guided by a myriad of local chemical messengers, hormones, and mechanical forces in the womb—all of which are devoid inside the spinning bioreactor. A more recent way to grow brain blobs is the guided method: scientists add a slew of “external patterning factors” at a very early stage of development, when stem cells first begin to choose their fate. These factors are basically biological fairy dust that push minibrain structures into a particular “pattern,” essentially sealing their fate.
Brain organoids grown this way are generally more consistent in cellular architecture once they mature. For example, many consistently develop the multi-striped pattern characteristic of the cerebral cortex—the outermost layer of the brain involved in sensation, reasoning, and other higher cognitive functions. But do they also resemble each other in their cellular makeup?
Reliable Brain Farming
The team first used both approaches to foster several dozen minibrains for half a year. They began with multiple types of stem cells from both male and female donors: induced pluripotent stem cells, which are skin cells returned to a youth-like stage, immortal human embryonic stem cells, and others.
They then carefully analyzed the resulting brainoids’ genetic makeup at multiple time points to track their growth. The team tapped an extremely powerful—and increasingly popular—tool called single-cell RNA sequencing, which provides invaluable insight into gene expression in every single cell.
In all, they parsed the genetic fingerprints of over 100,000 cells from 21 organoids, and matched those data to existing databases to tease out the cells’ identities. Finally, the team mapped out the distribution of each cell type in every analyzed organoid.
Unsurprisingly, those grown with the unguided method had cellular profiles all over the place. But with the guided approach—particularly, ones dubbed the “dorsally patterned” type—95 percent were “virtually indistinguishable” in their cellular compendium. What’s more, these minibrains also followed incredibly similar development trajectories, in that different cell types popped up at near-identical time points. Even their cellular origin didn’t matter: organoids grown from different stem cells were consistent in their final cellular inhabitants.
Conclusion? The embryo isn’t required for our brain to produce all its cellular diversity; it’s totally possible to reliably grow brainoids outside the womb.
The results are a huge boon for studying neurological diseases such as autism, epilepsy, and schizophrenia. Scientists believe that the root cause of these complex disorders lies somewhere in the tangled dance of fetal brain growth. So far, a clear cause has been elusive.
Using the guided “dorsally patterned” recipe, teams can now grow organoids from stem cells derived from patients, or genetically engineer pathological mutations to study their effects. Because the study proves minibrains made this way are remarkably similar, researchers will be able to nail down risk factors—and test potential treatments—without worrying about biological noise stemming from minibrain diversity.
Arlotta is already exploring possibilities. Using CRISPR, she plans to edit genes potentially linked to autism in stem cells, and grow them out as minibrains. Using the same technique, she can also make “control” organoids as a baseline for her experiments.
We can now “move much more swiftly towards concrete interventions, because they will direct us to the specific genetic features that give rise to the disease,” she said. “We will be able to ask far more precise questions about what goes wrong in the context of psychiatric illness.”
Jonah Reeder prepares a special protein shake that helps him manage a metabolic condition called phenylketonuria.
Instead of eating a typical breakfast every day, Jonah Reeder gulps down a special protein shake.
“The nutrients in it like to sit at the bottom, so I usually have to shake it up and get all the nutrients from the protein and everything,” says Reeder, 21, of Farmington, Utah, as he shakes a big plastic bottle.
Reeder was born with a rare genetic disorder called phenylketonuria, or PKU. If he eats meat, drinks milk or consumes other common sources of protein, toxic levels of the amino acid phenylalanine could build up in his body and damage his brain.
So Reeder gets his protein from the shake, which is rich in other amino acids, vitamins and proteins that don’t contain phenylalanine.
“It’s a really healthy drink,” Reeder says. “It’s basically protein, except without phenylalanine.”
But Reeder hopes a new approach for treating diseases could help people like him. The idea is to use bacteria that have been genetically modified to do what Reeder’s body can’t — get rid of phenylalanine.
“I’m really excited to help out and hopefully find a treatment for PKU,” Reeder said recently, as he prepared to volunteer for a study testing the modified bacteria.
The bacteria Reeder is helping test are part of a new field of medical research that has emerged from two realms of biomedical science. One is the study of the human microbiome, the microbes that inhabit our bodies. The other is synthetic biology, a field that looks at genetically engineering living organisms, including bacteria in the human gut.
“It’s a new world of being able to use synthetic biology to program microbes to treat diseases, which I believe is the future,” says Pamela Silver, a synthetic biologist at Harvard Medical School in Boston.
“Microbes are something that we as synthetic biologists see as highly engineer-able. We understand how to engineer microbes so it seems like the perfect interface between synthetic biology and health,” Silver says.
One company, ActoBio Therapeutics of Ghent, Belgium, has just started using genetically engineered microbes to try to treat Type 1 diabetes. Another one, Oragenics of Tampa, Fla., is testing a modified bacterium to treat mouth sores caused by cancer chemotherapy. And Osel of Mountain View, Calif., hopes engineered microbes could prevent HIV infections.
Reeder is helping test modified E. coli bacteria. While some types of E. coli can cause serious illness, the E. coli type being used in the study is found in the human gut.
“It is a naturally occurring probiotic bacteria,” says Caroline Kurtz, a scientist at Synlogic, a Cambridge, Mass., biotech company, which created the modified version of the organism.
“We can enhance its function by introducing genes [or] by changing genes that are there, and design the cells to either produce something or consume something that may be beneficial for a patient,” Kurtz says.
Synlogic has also engineered E. coli to rid life-threatening levels of ammonia from the bodies of people with cirrhosis of the liver.
“This is a really exciting new modality that allows us to think about therapies in a new way and really look at diseases in a whole new way: a living medicine that can respond to its environment,” Kurtz says.
Preliminary research involving mice and healthy adults published recently in the journal Science Translational Medicine indicates Synlogic’s E. coli are safe and may work. So the company is now testing them in patients with cirrhosis and PKU.
Reeder admits he was a little nervous when he first heard about all this.
“When you hear about E. coli you think: sickness, throwing up. So I was a little bit skeptical. I wasn’t sure what to think because I was going to be ingesting E. coli,” Reeder says.
But the more he learned about it, the more excited Reeder got about trying the engineered microbes.
“I think that’s very cool that they found a way to use a natural probiotic that’s found in the digestive tract to help the human body,” Reeder says.
Reeder spent the weekend in the clinic so doctors could monitor him closely and run tests as he ingested what normally would be dangerous amounts of protein. He then swallowed either the engineered E. coli or a placebo. He wasn’t told which.
“It was liquid solution. It tasted kind of like mint taffy. It was pretty sweet,” he says.
Reeder thinks he got the engineered microbes.
“I could immediately feel my cognitive abilities falling down after drinking the 20 grams of protein. And then I took the drug and I started feeling a lot better. I obtained more energy and my cognitive abilities got quicker,” he says.
“It was really cool to feel that. I could tell it was working. It was pretty cool,” Reeder says.
Much more research is needed to know whether genetically engineered microbes are safe and really work. But Synlogic hopes to report results from the cirrhosis and PKU studies later this year.
Before any of these experimental treatments could be used routinely, they would have to be reviewed and approved by the Food and Drug Administration. That’s a process that could take years.
New insights in biology show that food is informational and can directly impact and even control the expression of your genes. The implications of this discovery are profound, and have both a light and dark side in need of deeper exploration…
A new study published in the journal BMC Genetics entitled, “Plant miRNAs found in human circulating system provide evidence of cross kingdom RNAi,” reveals that powerful little diet derived nucleic acids known as microRNAs (miRNAs), from commonly consumed plants, are present within the human circulatory system in what appear to be physiologically significant quantities. MiRNAs are comprised of ~ 22 nucleotide single strand non-coding RNAs, which regulate protein coding gene expression by interfering with messenger RNA’s ability to transcribe DNA into protein. This is why miRNAs are sometimes called RNA interference molecules.
The study found,
“…abundant plant miRNAs sequences from 410 human plasma small RNA sequencing data sets. One particular plant miRNA miR2910, conserved in fruits and vegetables, was found to present in high relative amount in the plasma samples. This miRNA, with same 6mer and 7mer-A1 target seed sequences as hsa-miR-4259 and hsa-miR-4715-5p, was predicted to target human JAK-STAT signaling pathway gene SPRY4 and transcription regulation genes.”
This discovery has profound implications, as the human JAK-STAT signalling pathway has a wide range of potential downstream effects. In fact, JAK-STAT transmits information from extracellular chemical signals to the cell nucleus resulting in DNA transcription and expression of genes involved in immunity, differentiation, proliferation, apoptosis — all of which relate to cancer risk and oncogenesis. But this is just the tip of the miRNA iceberg. There have, in fact, been hundreds of these miRNAs identified in commonly consumed foods in the agrarian diet, and they appear to have the ability to match up with hundreds of human gene targets. The implications of this are profound, if not possibly devastating when it comes to GMO food technology.
It is now widely accepted among conventional biologists that miRNAs regulate most of the protein coding genes in mammals. In fact, the profound difference in complexity between higher life forms such as humans relative to, say, earthworms, is attributable to the higher level of RNA complexity within the so-called ‘dark matter of the genome’ (the ~ 98.5% of the human genome that does not code for proteins).
But what research like this brings to the table is the even more provocative possibility that our genetic and epigenetic wellbeing may be wholly dependent on miRNAs existing outside of us within the gene-regulatory miRNAs embedded within our diet.
Can you imagine the difference between an evolutionarily conserved ancestral diet and a modern one comprised of synthetic components and highly processed GMO cereal grasses?
The New Epigenetic/Nutritional Paradigm: Cross-Kingdom Communication
The idea that the plants and animals we eat contribute to modulating the expression of our genome is known as cross-kingdom or inter-species genentic communication, and represents a significant departure from the classical view that the genetic infrastructure of species were closed off, hermetically sealed within the cell nucleus, and could not be accessed epigenetically from the outside in. We’ve moved from this atomistic, monadistic view to an open access one, where miRNAs operate like software upon the hardwired protein-coding sequences within a species’ genome, making for a much more complex and interdependent web of relationships, reminiscent of the Gaian concept of a biospheric interconnectivity between all the biotic elements of the Earth. As I discuss in another article,
“…this more “open access” model would permit species to alter and affect another’s phenotype in real-time, along with potentially altering its long-term evolutionary trajectory by affecting epigenetic inheritance patterns. This speaks to a co-evolutionary and co-operative model, with all areas of the tree of life, co-developing in a highly complex and seemingly highly intelligent, carefully orchestrated manner.”
And so, if plant derived miRNAs can survive cooking and digestion, as appears to be the case, and can accumulate in physiologically significant quantities, they will therefore alter gene expression, introducing the novel concept that mammalian genomes may have, in fact, evolved to outsource some of their regulation to nutrigenomic dimensions within their dietary milieux.
This, of course, has profound implications, such as validating the concept that an evolutionarily appropriate diet — e.g. Paleo diet — would help to assure the optimal expression of the human genome. Conversely, the use of RNA interference technology by biotech corporations, such as Monsanto/Dow’s newly EPA approved RNAi corn, could have biologically devastating consequences to the health and wellbeing of those fed or exposed to its altered miRNA profiles. To learn more about this concerning possibility, read (and please share) my report: The GMO Agenda Takes a Menacing Leap Forward with EPA’s Silent Approval of Monsanto/Dow’s RNAi Corn
Engineered microorganisms churn out THC, CBD and rarer, less-understood cannabis cousins
If you had to pick a favorite microbe, a good candidate would be Saccharomyces cerevisiae, better known as brewer’s yeast, which transforms grape juice into wine, grain mash into beer and dough into bread. Over the past few decades scientists have hacked the yeast’s genome to make it produce less delectable but arguably more important substances, including hormones like insulin and drugs like opioids. Now it is churning out cannabinoids, the compounds found in marijuana.
Researchers led by Jay Keasling, a professor of chemical engineering and bioengineering at the University of California, Berkeley, have genetically modified brewer’s yeast to produce two of the most common cannabinoids, tetrahydrocannabinol (THC) and cannabidiol (CBD). They claim their method could also produce microorganisms capable of making any other naturally occurring cannabinoid as well as some brand-new varieties. Certain cannabinoids have potential as treatments for a variety of disorders, but require more research to separate hype from medical reality.
In the new study published Wednesday in Nature, scientists transferred known gene sequences that control metabolic pathways in cannabis plants into yeast. The resulting microorganisms can turn a sugar called galactose into intermediate chemicals, and use those chemicals to synthesize cannabigerolic acid (CBGa)—the so-called mother cannabinoid that can develop into several other compounds. Finally, each individual strain of yeast turns CBGa into a different cannabinoid. “The yeast that produces THC is different from the yeast that produces CBD, but they only differ by one gene—and that’s the last gene in the pathway that takes CBGa and turns it into CBD or THC,” Keasling says. “The beauty of this technology is that you can swap these out for a rare cannabinoid.”
Marijuana contains more than 100 different cannabinoids, but most of them are at much lower concentrations than CBD and THC. Because plants yield very small amounts of the rarer substances, they are more expensive to produce. Even when researchers successfully extract them, the compounds are often contaminated with traces of their more common cousins. Yeast could produce purer versions of these cannabinoids, bringing the price of rare varieties to the same level as more popular ones. “It’s a platform for producing all of the cannabinoids that are currently thought to exist in cannabis as well as all these unnatural ones that you’d never find in any organism,” Keasling says. What makes some of these “unnatural”? Normally, marijuana plants incorporate a chemical called hexanoic acid (which humans use as a cheap food additive) into cannabinoids. When Keasling’s team added different chemicals into their yeast’s sugary diet, the genetically modified microbes incorporated those substances instead of the hexanoic acid, resulting in new, never-before-seen compounds.
Putting Cannabinoids to the Test
“There might be a blockbuster drug or two in some of those rare ones or unnatural” cannabinoids, Keasling says. But uncovering the potential medical applications will take a lot more research—and scientists are already busy studying the effects of the more famous cannabinoids. For example, CBD shows promise for treating problems such as epilepsy, PTSD and addiction without producing the high that psychoactive THC does. The hype around CBD, however, has built up faster than the scientific research on it; proponents have made unsubstantiated claims of its ability to treat just about anything, from eczema to cancer. This promotion has gone mainstream, with coffee shops putting CBD on the menu as a supposed anxiety treatment and online vendors selling inaccurately labeled extracts.
To find the compounds’ true applications, researchers must test how cannabinoids affect humans. That is a problem this new biosynthesis method may not solve, however. “It doesn’t matter if CBD or any cannabinoids come from a plant or if they’re made synthetically or if they come from yeast. If the end product is still a banned substance, it doesn’t increase accessibility,” says Yasmin Hurd, director of the Addiction Institute at Icahn School of Medicine at Mount Sinai in New York City, who was not involved in the new study. The Drug Enforcement Administration classifies marijuana-derived substances like CBD as Schedule I drugs—a category that includes heroin. This gets in the way of researchers like Hurd, who studies how CBD impacts craving and anxiety—factors that contribute to opioid addiction. “It’s really important to have more research done—it’s only then that we will be able to see whether CBD or other specific cannabinoids that people are making claims about…are able to treat these symptoms and disorders,” she says.
Keasling agrees that, despite looser state laws, federal restrictions on testing cannabis make research difficult—“and there’s no way to get around that until [the] law changes,” he says. “But what this method can do is provide some of these very rare cannabinoids that you’d never be able to extract out of cannabis, because they’re produced in such small quantities. And who knows—one of those might be better than CBD or THC.“
A Growing Industry
Whether or not they help research, yeast-made cannabinoids will certainly have a commercial impact. Keasling estimates his method could produce cannabinoids at a cost equal to or lower than that required by agricultural marijuana production. He founded the company Demetrix to license his new technology from Berkeley and develop commercial cannabinoid production. And he has plenty of competition. Last year biotechnology start-ups Librede and Gingko Bioworks announced, respectively, a patent for producing CBD from yeast and a multimillion-dollar partnership with cannabis company Cronos Group. Demetrix CEO Jeff Ubersax estimates that about 15 to 20 other companies are competing to turn yeast cells into little cannabinoid factories.
Yet Keasling claims his team is the first to develop a process that relies only on sugar—his yeast can make cannabinoids from galactose alone without requiring additional, more expensive ingredients. Furthermore, Ubersax says the new yeast produces cannabinoids at a rate several orders of magnitude higher than that of other strains. “Prior work had identified the basic parts you need but was producing them at a very low level,” he says, comparing the output with that of a compact car engine. Keasling, he says, “identified a new DNA segment that’s more like a jet engine.”
Still, this explosion of start-ups will not help investigators like Hurd any more than the rise of CBD lattes has. “The problem is that companies are not putting any money investments into research,” she says. They are not contributing anything to the advancement of knowledge, she adds. Without further study the federal government will not sanction more uses for cannabinoids—the U.S. Food and Drug Administration has only approved one drug containing substances derived from marijuana (the epilepsy medication Epidiolex, which includes CBD). Hurd has an idea to break this cycle. “Tax the companies and put that tax money toward research,” she says. “For companies that want to make a lot of money in CBD, say, ‘Okay, this is your contribution.’ And let’s just get the research done so [the federal government] can approve more.”
Interest in a powerful DNA editing tool called CRISPR has revealed that bacteria are far more sophisticated than anyone imagined.
On a November evening last year, Jennifer Doudna put on a stylish black evening gown and headed to Hangar One, a building at NASA’s Ames Research Center that was constructed in 1932 to house dirigibles. Under the looming arches of the hangar, Doudna mingled with celebrities like Benedict Cumberbatch, Cameron Diaz and Jon Hamm before receiving the 2015 Breakthrough Prize in life sciences, an award sponsored by Mark Zuckerberg and other tech billionaires. Doudna, a biochemist at the University of California, Berkeley, and her collaborator, Emmanuelle Charpentier of the Helmholtz Centre for Infection Research in Germany, each received $3 million for their invention of a potentially revolutionary tool for editing DNA known as CRISPR.
Doudna was not a gray-haired emerita being celebrated for work she did back when dirigibles ruled the sky. It was only in 2012 that Doudna, Charpentier and their colleagues offered the first demonstration of CRISPR’s potential. They crafted molecules that could enter a microbe and precisely snip its DNA at a location of the researchers’ choosing. In January 2013, the scientists went one step further: They cut out a particular piece of DNA in human cells and replaced it with another one.
In the same month, separate teams of scientists at Harvard University and the Broad Institute reported similar success with the gene-editing tool. A scientific stampede commenced, and in just the past two years, researchers have performed hundreds of experiments on CRISPR. Their results hint that the technique may fundamentally change both medicine and agriculture.
Some scientists have repaired defective DNA in mice, for example, curing them of genetic disorders. Plant scientists have used CRISPR to edit genes in crops, raising hopes that they can engineer a better food supply. Some researchers are trying to rewrite the genomes of elephants, with the ultimate goal of re-creating a woolly mammoth. Writing last year in the journal Reproductive Biology and Endocrinology, Motoko Araki and Tetsuya Ishii of Hokkaido University in Japan predicted that doctors will be able to use CRISPR to alter the genes of human embryos “in the immediate future.”
Thanks to the speed of CRISPR research, the accolades have come quickly. Last year MIT Technology Reviewcalled CRISPR “the biggest biotech discovery of the century.” The Breakthrough Prize is just one of several prominent awards Doudna has won in recent months for her work on CRISPR; National Public Radio recently reported whispers of a possible Nobel in her future.
Even the pharmaceutical industry, which is often slow to embrace new scientific advances, is rushing to get in on the act. New companies developing CRISPR-based medicine are opening their doors. In January, the pharmaceutical giant Novartis announced that it would be using Doudna’s CRISPR technology for its research into cancer treatments. It plans to edit the genes of immune cells so that they will attack tumors.
But amid all the black-tie galas and patent filings, it’s easy to overlook the most important fact about CRISPR: Nobody actually invented it.
Doudna and other researchers did not pluck the molecules they use for gene editing from thin air. In fact, they stumbled across the CRISPR molecules in nature. Microbes have been using them to edit their own DNA for millions of years, and today they continue to do so all over the planet, from the bottom of the sea to the recesses of our own bodies.
We’ve barely begun to understand how CRISPR works in the natural world. Microbes use it as a sophisticated immune system, allowing them to learn to recognize their enemies. Now scientists are discovering that microbes use CRISPR for other jobs as well. The natural history of CRISPR poses many questions to scientists, for which they don’t have very good answers yet. But it also holds great promise. Doudna and her colleagues harnessed one type of CRISPR, but scientists are finding a vast menagerie of different types. Tapping that diversity could lead to more effective gene editing technology, or open the way to applications no one has thought of yet.
“You can imagine that many labs — including our own — are busily looking at other variants and how they work,” Doudna said. “So stay tuned.”
A Repeat Mystery
The scientists who discovered CRISPR had no way of knowing that they had discovered something so revolutionary. They didn’t even understand what they had found. In 1987, Yoshizumi Ishino and colleagues at Osaka University in Japan published the sequence of a gene called iap belonging to the gut microbe E. coli. To better understand how the gene worked, the scientists also sequenced some of the DNA surrounding it. They hoped to find spots where proteins landed, turning iap on and off. But instead of a switch, the scientists found something incomprehensible.
Near the iap gene lay five identical segments of DNA. DNA is made up of building blocks called bases, and the five segments were each composed of the same 29 bases. These repeat sequences were separated from each other by 32-base blocks of DNA, called spacers. Unlike the repeat sequences, each of the spacers had a unique sequence.
This peculiar genetic sandwich didn’t look like anything biologists had found before. When the Japanese researchers published their results, they could only shrug. “The biological significance of these sequences is not known,” they wrote.
It was hard to know at the time if the sequences were unique to E. coli, because microbiologists only had crude techniques for deciphering DNA. But in the 1990s, technological advances allowed them to speed up their sequencing. By the end of the decade, microbiologists could scoop up seawater or soil and quickly sequence much of the DNA in the sample. This technique — called metagenomics — revealed those strange genetic sandwiches in a staggering number of species of microbes. They became so common that scientists needed a name to talk about them, even if they still didn’t know what the sequences were for. In 2002, Ruud Jansen of Utrecht University in the Netherlands and colleagues dubbed these sandwiches “clustered regularly interspaced short palindromic repeats” — CRISPR for short.
Jansen’s team noticed something else about CRISPR sequences: They were always accompanied by a collection of genes nearby. They called these genes Cas genes, for CRISPR-associated genes. The genes encoded enzymes that could cut DNA, but no one could say why they did so, or why they always sat next to the CRISPR sequence.
Three years later, three teams of scientists independently noticed something odd about CRISPR spacers. They looked a lot like the DNA of viruses.
At the time, Koonin, an evolutionary biologist at the National Center for Biotechnology Information in Bethesda, Md., had been puzzling over CRISPR and Cas genes for a few years. As soon as he learned of the discovery of bits of virus DNA in CRISPR spacers, he realized that microbes were using CRISPR as a weapon against viruses.
Koonin knew that microbes are not passive victims of virus attacks. They have several lines of defense. Koonin thought that CRISPR and Cas enzymes provide one more. In Koonin’s hypothesis, bacteria use Cas enzymes to grab fragments of viral DNA. They then insert the virus fragments into their own CRISPR sequences. Later, when another virus comes along, the bacteria can use the CRISPR sequence as a cheat sheet to recognize the invader.
Scientists didn’t know enough about the function of CRISPR and Cas enzymes for Koonin to make a detailed hypothesis. But his thinking was provocative enough for a microbiologist named Rodolphe Barrangou to test it. To Barrangou, Koonin’s idea was not just fascinating, but potentially a huge deal for his employer at the time, the yogurt maker Danisco. Danisco depended on bacteria to convert milk into yogurt, and sometimes entire cultures would be lost to outbreaks of bacteria-killing viruses. Now Koonin was suggesting that bacteria could use CRISPR as a weapon against these enemies.
To test Koonin’s hypothesis, Barrangou and his colleagues infected the milk-fermenting microbe Streptococcus thermophilus with two strains of viruses. The viruses killed many of the bacteria, but some survived. When those resistant bacteria multiplied, their descendants turned out to be resistant too. Some genetic change had occurred. Barrangou and his colleagues found that the bacteria had stuffed DNA fragments from the two viruses into their spacers. When the scientists chopped out the new spacers, the bacteria lost their resistance.
Barrangou, now an associate professor at North Carolina State University, said that this discovery led many manufacturers to select for customized CRISPR sequences in their cultures, so that the bacteria could withstand virus outbreaks. “If you’ve eaten yogurt or cheese, chances are you’ve eaten CRISPR-ized cells,” he said.
Cut and Paste
As CRISPR started to give up its secrets, Doudna got curious. She had already made a name for herself as an expert on RNA, a single-stranded cousin to DNA. Originally, scientists had seen RNA’s main job as a messenger. Cells would make a copy of a gene using RNA, and then use that messenger RNA as a template for building a protein. But Doudna and other scientists illuminated many other jobs that RNA can do, such as acting as sensors or controlling the activity of genes.
In 2007, Blake Wiedenheft joined Doudna’s lab as a postdoctoral researcher, eager to study the structure of Cas enzymes to understand how they worked. Doudna agreed to the plan — not because she thought CRISPR had any practical value, but just because she thought the chemistry might be cool. “You’re not trying to get to a particular goal, except understanding,” she said.
As Wiedenheft, Doudna and their colleagues figured out the structure of Cas enzymes, they began to see how the molecules worked together as a system. When a virus invades a microbe, the host cell grabs a little of the virus’s genetic material, cuts open its own DNA, and inserts the piece of virus DNA into a spacer.
As the CRISPR region fills with virus DNA, it becomes a molecular most-wanted gallery, representing the enemies the microbe has encountered. The microbe can then use this viral DNA to turn Cas enzymes into precision-guided weapons. The microbe copies the genetic material in each spacer into an RNA molecule. Cas enzymes then take up one of the RNA molecules and cradle it. Together, the viral RNA and the Cas enzymes drift through the cell. If they encounter genetic material from a virus that matches the CRISPR RNA, the RNA latches on tightly. The Cas enzymes then chop the DNA in two, preventing the virus from replicating.
This video illustrates how CRISPR and Cas9 can help microbes fight viruses and how researchers might use that system to edit human genes.
As CRISPR’s biology emerged, it began to make other microbial defenses look downright primitive. Using CRISPR, microbes could, in effect, program their enzymes to seek out any short sequence of DNA and attack it exclusively.
“Once we understood it as a programmable DNA-cutting enzyme, there was an interesting transition,” Doudna said. She and her colleagues realized there might be a very practical use for CRISPR. Doudna recalls thinking, “Oh my gosh, this could be a tool.”
It wasn’t the first time a scientist had borrowed a trick from microbes to build a tool. Some microbes defend themselves from invasion by using molecules known as restriction enzymes. The enzymes chop up any DNA that isn’t protected by molecular shields. The microbes shield their own genes, and then attack the naked DNA of viruses and other parasites. In the 1970s, molecular biologists figured out how to use restriction enzymes to cut DNA, giving birth to the modern biotechnology industry.
In the decades that followed, genetic engineering improved tremendously, but it couldn’t escape a fundamental shortcoming: Restriction enzymes did not evolve to make precise cuts — only to shred foreign DNA. As a result, scientists who used restriction enzymes for biotechnology had little control over where their enzymes cut open DNA.
The CRISPR-Cas system, Doudna and her colleagues realized, had already evolved to exert just that sort of control.
To create a DNA-cutting tool, Doudna and her colleagues picked out the CRISPR-Cas system from Streptococcus pyogenes, the bacteria that cause strep throat. It was a system they already understood fairly well, having worked out the function of its main enzyme, called Cas9. Doudna and her colleagues figured out how to supply Cas9 with an RNA molecule that matched a sequence of DNA they wanted to cut. The RNA molecule then guided Cas9 along the DNA to the target site, and then the enzyme made its incision.
Using two Cas9 enzymes, the scientists could make a pair of snips, chopping out any segment of DNA they wanted. They could then coax a cell to stitch a new gene into the open space. Doudna and her colleagues thus invented a biological version of find-and-replace — one that could work in virtually any species they chose to work on.
As important as these results were, microbiologists were also grappling with even more profound implications of CRISPR. It showed them that microbes had capabilities no one had imagined before.
Before the discovery of CRISPR, all the defenses that microbes were known to use against viruses were simple, one-size-fits-all strategies. Restriction enzymes, for example, will destroy any piece of unprotected DNA. Scientists refer to this style of defense as innate immunity. We have innate immunity, too, but on top of that, we also use an entirely different immune system to fight pathogens: one that learns about our enemies.
This so-called adaptive immune system is organized around a special set of immune cells that swallow up pathogens and then present fragments of them, called antigens, to other immune cells. If an immune cell binds tightly to an antigen, the cell multiplies. The process of division adds some random changes to the cell’s antigen receptor genes. In a few cases, the changes alter the receptor in a way that lets it grab the antigen even more tightly. Immune cells with the improved receptor then multiply even more.
This cycle results in an army of immune cells with receptors that can bind quickly and tightly to a particular type of pathogen, making them into precise assassins. Other immune cells produce antibodies that can also grab onto the antigens and help kill the pathogen. It takes a few days for the adaptive immune system to learn to recognize the measles virus, for instance, and wipe it out. But once the infection is over, we can hold onto these immunological memories. A few immune cells tailored to measles stay with us for our lifetime, ready to attack again.
CRISPR, microbiologists realized, is also an adaptive immune system. It lets microbes learn the signatures of new viruses and remember them. And while we need a complex network of different cell types and signals to learn to recognize pathogens, a single-celled microbe has all the equipment necessary to learn the same lesson on its own.
A New Kind of Evolution
CRISPR is an impressive adaptive immune system for another reason: Its lessons can be inherited. People can’t pass down genes for antibodies to their children because only immune cells develop them. There’s no way for that information to get into eggs or sperm. As a result, children have to start learning about their invisible enemies pretty much from scratch.
CRISPR is different. Since microbes are single-celled organisms, the DNA they alter to fight viruses is the same DNA they pass down to their descendants. In other words, the experiences that these organisms have alter their genes, and that change is inherited by future generations.
For students of the history of biology, this kind of heredity echoes a largely discredited theory promoted by the naturalist Jean-Baptiste Lamarck in the early 19th century. Lamarck argued for the inheritance of acquired traits. To illustrate his theory, he had readers imagine a giraffe gaining a long neck by striving to reach high branches to feed on. A nervous fluid, he believed, stretched out its neck, making it easier for the giraffe to reach the branches. It then passed down its lengthened neck to its descendants.
The advent of genetics seemed to crush this idea. There didn’t appear to be any way for experiences to alter the genes that organisms passed down to their offspring. But CRISPR revealed that microbes rewrite their DNA with information about their enemies — information that Barrangou showed could make the difference between life and death for their descendants.
Did this mean that CRISPR meets the requirements for Lamarckian inheritance? “In my humble opinion, it does,” said Koonin.
But how did microbes develop these abilities? Ever since microbiologists began discovering CRISPR-Cas systems in different species, Koonin and his colleagues have been reconstructing the systems’ evolution. CRISPR-Cas systems use a huge number of different enzymes, but all of them have one enzyme in common, called Cas1. The job of this universal enzyme is to grab incoming virus DNA and insert it in CRISPR spacers. Recently, Koonin and his colleagues discovered what may be the origin of Cas1 enzymes.
Along with their own genes, microbes carry stretches of DNA called mobile elements that act like parasites. The mobile elements contain genes for enzymes that exist solely to make new copies of their own DNA, cut open their host’s genome, and insert the new copy. Sometimes mobile elements can jump from one host to another, either by hitching a ride with a virus or by other means, and spread through their new host’s genome.
Koonin and his colleagues discovered that one group of mobile elements, called casposons, makes enzymes that are pretty much identical to Cas1. In a new paper in Nature Reviews Genetics, Koonin and Mart Krupovic of the Pasteur Institute in Paris argue that the CRISPR-Cas system got its start when mutations transformed casposons from enemies into friends. Their DNA-cutting enzymes became domesticated, taking on a new function: to store captured virus DNA as part of an immune defense.
While CRISPR may have had a single origin, it has blossomed into a tremendous diversity of molecules. Koonin is convinced that viruses are responsible for this. Once they faced CRISPR’s powerful, precise defense, the viruses evolved evasions. Their genes changed sequence so that CRISPR couldn’t latch onto them easily. And the viruses also evolved molecules that could block the Cas enzymes. The microbes responded by evolving in their turn. They acquired new strategies for using CRISPR that the viruses couldn’t fight. Over many thousands of years, in other words, evolution behaved like a natural laboratory, coming up with new recipes for altering DNA.
The Hidden Truth
To Konstantin Severinov, who holds joint appointments at Rutgers University and the Skolkovo Institute of Science and Technology in Russia, these explanations for CRISPR may turn out to be true, but they barely begin to account for its full mystery. In fact, Severinov questions whether fighting viruses is the chief function of CRISPR. “The immune function may be a red herring,” he said.
Severinov’s doubts stem from his research on the spacers of E. coli. He and other researchers have amassed a database of tens of thousands of E. coli spacers, but only a handful of them match any virus known to infect E. coli. You can’t blame this dearth on our ignorance of E. coli or its viruses, Severinov argues, because they’ve been the workhorses of molecular biology for a century. “That’s kind of mind-boggling,” he said.
It’s possible that the spacers came from viruses, but viruses that disappeared thousands of years ago. The microbes kept holding onto the spacers even when they no longer had to face these enemies. Instead, they used CRISPR for other tasks. Severinov speculates that a CRISPR sequence might act as a kind of genetic bar code. Bacteria that shared the same bar code could recognize each other as relatives and cooperate, while fighting off unrelated populations of bacteria.
But Severinov wouldn’t be surprised if CRISPR also carries out other jobs. Recent experiments have shown that some bacteria use CRISPR to silence their own genes, instead of seeking out the genes of enemies. By silencing their genes, the bacteria stop making molecules on their surface that are easily detected by our immune system. Without this CRISPR cloaking system, the bacteria would blow their cover and get killed.
“This is a fairly versatile system that can be used for different things,” Severinov said, and the balance of all those things may differ from system to system and from species to species.
If scientists can get a better understanding of how CRISPR works in nature, they may gather more of the raw ingredients for technological innovations. To create a new way to edit DNA, Doudna and her colleagues exploited the CRISPR-Cas system from a single species of bacteria, Streptococcus pyogenes. There’s no reason to assume that it’s the best system for that application. At Editas, a company based in Cambridge, Massachusetts, scientists have been investigating the Cas9 enzyme made by another species of bacteria, Staphylococcus aureus. In January, Editas scientists reported that it’s about as efficient at cutting DNA as Cas9 from Streptococcus pyogenes. But it also has some potential advantages, including its small size, which may make it easier to deliver into cells.
To Koonin, these discoveries are just baby steps into the ocean of CRISPR diversity. Scientists are now working out the structure of distantly related versions of Cas9 that seem to behave very differently from the ones we’re now familiar with. “Who knows whether this thing could become even a better tool?” Koonin said.
And as scientists discover more tasks that CRISPR accomplishes in nature, they may be able to mimic those functions, too. Doudna is curious about using CRISPR as a diagnostic tool, searching cells for cancerous mutations, for example. “It’s seek and detect, not seek and destroy,” she said. But having been surprised by CRISPR before, Doudna expects the biggest benefits from these molecules to surprise us yet again. “It makes you wonder what else is out there,” she said.
Jennifer Doudna, one of CRISPR’s primary innovators, stays optimistic about how the gene-editing tool will continue to empower basic biological understanding.
No one needs to remind Jennifer Doudna about the power of CRISPR, the precision genome-editing technology she codeveloped. CRISPR “gives us a way to ultimately control the evolution of any organism — including ourselves. It is a profound thing. Human beings have now learned enough about our own genetic code that we can change it at will,” she said. “It’s kind of crazy to think about.”
That’s why when reports emerged last November that the scientist He Jiankui of the Southern University of Science and Technology in Shenzhen, China, had used CRISPR to alter the DNA of twin baby girls — crossing a line that genetic engineers had respected for decades and reaffirmed in 2015 — Doudna was quick to speak out. Describing herself to the media as “horrified and stunned,” she criticized his actions as risky, premature and unnecessary, given the absence of pressing medical need for the children to be modified experimentally. She encouraged the international scientific community to develop better guidelines for permissible genome editing in humans.
CRISPR technology makes genome editing temptingly simple because it allows scientists to cut and edit sequences of DNA in any species, including humans, at will. It was inspired by a long-overlooked defense mechanism with which many bacteria fend off viruses: By inserting fragments of viral DNA into specialized structures in their own genome (the “clustered regularly interspaced short palindromic repeats” that give CRISPR its name), bacteria provide their daughter cells with a way to recognize and quickly rebuff future invasions by similar viruses. Doudna and Emmanuelle Charpentier of the Max Planck Institute for Infection Biology in Berlin showed in 2012 that the bacterial system could be adapted as an editing tool. (Several other scientists, including Feng Zhang of the Broad Institute and Virginijus Šikšnys of Vilnius University are also credited with contributing to CRISPR’s development, and multiple patent lawsuits surround the ownership of the intellectual property.)
But although Doudna argues for caution when contemplating changes that could be passed down for generations, she remains a forceful advocate for the potential of CRISPR in basic research, as well as its medical and biotech applications. “I think when you understand how things work, you can apply them more effectively. And once you apply them, you invariably uncover things that you didn’t understand about the fundamental biology of that system,” Doudna said. “I love that kind of interplay.”
Quanta Magazine sat down with Doudna, a professor of chemistry and molecular and cell biology at the University of California, Berkeley, to discuss how CRISPR is furthering basic biology research. That interview and subsequent exchanges have been condensed and edited for clarity.
When CRISPR is discussed in the public, it’s often in regard to how it will be used to cure and treat disease. How do you think CRISPR has and will further basic biology research?
I’ll give you two examples that I think are fun because they illustrate some of the creative things people are doing now that weren’t possible in the past. One of them is a project to investigate the origins of bipedalism. This project involves comparing the genetics of two types of rodents — a standard quadruped mouse and a rodent called a jerboa that hops on its hind legs, so it’s bipedal.
What if I start putting genes from the bipedal rodent into the other rodent? Can I eventually make a bipedal field mouse? This is the kind of project that’s possible now with CRISPR.
The other CRISPR experiment I want to highlight explores the content of the Neanderthal genome. A lot of us now know that we have a little bit of Neanderthal DNA in our background, but what were the real differences between modern humans and Neanderthals? Why did Neanderthals go extinct, and what can that tell us about our own evolution?
How do you explore that?
An experiment that’s now underway is taking genes from the Neanderthal genome and putting them into human cells that are cultured in the lab in the form of brain organoids. Organoids are balls of tissue that form organlike structures in a laboratory dish. I don’t want to say they’re like little brains, but these organoids have some properties of collections of neurons found in the brain.
The question then is to ask, if you start introducing Neanderthal specific genes into these human brain organoids, what happens? What kinds of changes do we see physiologically in those cells and in those balls of tissue? Can we learn something about the genetics of neuronal development in Neanderthals that might be different from what happens in humans?
It’s the early days of this research, and an organoid is not the same thing as a brain, so there’s going to have to be some interpretations of what the data mean, but I think that’s the kind of experiment that couldn’t have been done before.
For the most part, we didn’t have a way to introduce changes to genomes precisely. The way that gene therapy was done originally was using viruses that integrate into human DNA, but the viruses integrate where they want to go, not necessarily where you want to make a change to the DNA.
Earlier technologies for gene editing were also very difficult to use in many settings. They required a lot of legwork to develop, like engineering particular proteins for each desired change to a genome. Certainly, it was difficult with those technologies to make more than one change at a time. With CRISPR, experimenters can change multiple genes in a genome in one shot.
Do you think that the medical applications of CRISPR in themselves can inform basic science?
For sure. CRISPR technology has been widely adopted by all kinds of scientists, including people like me. I was never doing anything with genome editing before CRISPR came along.
In my lab we’ve had a project over the last few years working on Huntington’s chorea, a degenerative neurological disease. The mutation that causes the disease is a single codon — three base pairs in the DNA — that gets repeated many times. If the codon gets repeated too many times, it leads to a defective protein that causes this disease. That’s been known for a long time, but the challenge was, how do you fix it?
We’ve been working on a way to deliver the CRISPR into mouse neuronal cells to make the necessary edits. But one of the curious things that’s come out of that line of work is that we found that only neuronal cells in the mouse brain were getting edited, not [the supportive glial] cells called astrocytes.
These cells are a lot smaller, so it could be that they don’t have enough surface area to take up the CRISPR protein efficiently. Or maybe they don’t respond to DNA cutting and editing in the same way as other cells.
So not all the cells in the brain respond to the CRISPR treatment the same way.
We also found that, when we inject CRISPR molecules into one place in the mouse brain, we see cells that are a fair distance away from the injection site also get edited. That was a surprise because it suggests there’s some way of trafficking molecules through the brain to areas that are not right next door to where the needle goes in.
Is there some mechanism for molecular trafficking in the brain that hasn’t been appreciated? That’s a very fundamental question in biology. That takes us back to asking, “Gee, how does the brain work?” Now we’re exploring answers to these questions.
I have to say that this example from my lab is emblematic of what’s going on in a variety of labs. CRISPR enables very applied experiments, but these experiments raise very fundamental questions that you have to go back and address.
How pervasive do you think the use of CRISPR will become in biology? It already seems to be everywhere in biomedical labs. Do you think there might be applications in fields like ecology, for example?
Absolutely. It depends on your definition of ecology, but some of the early adopters of the CRISPR technology were people who were trying to understand the genetics of butterfly development. Having a tool that allowed the manipulation of genes in non-model organisms — organisms that scientists haven’t been working on for decades in the laboratory but instead collected in the wild — opens up the possibility of real experimentation in a way that previously was not possible.
Effectively any organism becomes a model organism — a genetically tractable system for doing experiments. We joke in the lab that we’re turning humans into yeast: In the past, you had to do experiments in yeast or fruit flies and then infer what those data meant about human cells. You couldn’t easily manipulate human cells genetically to understand genetic function. Now, with CRISPR, you can.
CRISPR was first discovered as a defense mechanism that bacteria use against viruses. Do you think other new research tools might come from other discoveries about bacteria?
I suspect so. If you look at the technologies that have come along over the last several decades — polymerase chain reaction for amplifying DNA, molecular cloning — they all came about from studying how microbes fight off viruses or respond to stimuli.
We also know very little about the bacterial world right now. There’s a huge number of organisms that have never been identified by scientists or cultivated in the laboratory, and they surely have interesting biology associated with their lifestyles. As more of those organisms are studied and identified, I have no doubt that we’ll find things that will lend themselves to new technologies.
Are there any particular enigmas in bacteria that you have a hunch might lead to some sort of research tool?
That’s always hard to predict. That being said, I’ll give you an example of an interesting phenomenon: the discovery of this new category of bacteria that are incredibly small. It’s a whole new phylum of organisms — they’re currently called the candidate phyla radiation (CPR) bacteria. They almost challenge the notion of what’s a cell and what’s a virus.
A lot of these organisms probably grow symbiotically with other bugs, sharing important molecules, maybe even the building blocks of DNA, RNA and proteins. But how do they import molecules? How do they control their environment so that other kinds of bacteria don’t overgrow and crowd them out?
These are all unanswered questions. We don’t understand anything about their fundamental biology in a molecular sense. Will answers to these questions lead to a new technology? I don’t know, but it’s certainly going to lead to interesting biology.
So, the place to look for new research tools might be organisms that are atypical, so to speak?
But how do you define atypical? There’s this old Steve Forbert song: “It’s often said that life is strange … but compared to what?”
These tiny CPR bacteria are the ones in which you and Jillian Banfield of the University of California, Berkeley recently found new Cas enzymes [for cutting strands of DNA] that could be used with CRISPR technology, aren’t they? What makes those Cas enzymes potentially so interesting and useful?
One of the new enzymes we identified is called “CasX.” It’s particularly interesting because it seems to work quite differently from its cousin Cas9, the enzyme that many conventional bacteria use in their CRISPR defenses and that’s commonly used in CRISPR technology. But a few core ingredients are the same. This gives us insight into the basic recipe for CRISPR cutting proteins. The more we understand these proteins, the better we can engineer them. CasX is also appealing because it’s much smaller than Cas9, which might make it easier to slip into cells for therapeutic genome editing.
There have also been other new spinoff technologies developing out of CRISPR-Cas9, like CRISPR-GO, DNA imaging and anti-CRISPR. How might they help basic biology?
So let’s just go through those. CRISPR-GO is this clever way of using CRISPR enzymes to bring particular parts of the genome into physical proximity. There’s evidence that when genes are being expressed together in cells, they’re often brought together physically to the same location in the cells, and that can fundamentally affect the levels of proteins that are produced from certain genes. What CRISPR-GO does is provide a technology for doing that kind of physical tethering, except now the scientists can control it rather than the cell controlling it. I think that creates an opportunity to start dissecting the relationship between the 3D architecture of the genome and the communication between genes, and the resulting levels of proteins or RNA molecules that are made from those genes. So that’s exciting. It’s something that, again, really hasn’t been possible before, to control the 3D architecture of chromosomes and ask how that affects the output from the genome.
You mentioned DNA imaging. The idea there is what’s being referred to as “chromosome painting,” where you can program the CRISPR-Cas9 protein to bind and basically sit for extended periods of time at certain places in the DNA. You can decorate the CRISPR-Cas9 protein with different colors of dyes to light up a particular gene or section of a genome, even an entire chromosome, by just tiling it with these little RNA-protein complexes. So it’s a method for imaging.
In the case of anti-CRISPR, these are teeny tiny natural proteins involved in regulating CRISPR systems. You can imagine that in bacteria that are getting infected by viruses, over time viruses have evolved ways of avoiding being taken out by CRISPRs, and one of the ways they do that is using these little inhibitors called anti-CRISPRs. There’s interest in these because of the potential to control gene-editing outcomes — using these kinds of proteins to turn off gene-editing proteins in cells to protect the genome from being modified in unintended ways. There’s a whole line of research now that’s taken off to look at natural regulators and inhibitors of CRISPR pathways and ask whether those can be harnessed for technology purposes.
Could the development of anti-CRISPR quell fears about genome editing in humans or other organisms, if we had an off switch to throw if CRISPR-Cas9 wasn’t working as intended?
That’s exactly what people are thinking about. In fact, there’s a whole program funded by DARPA (the U.S. Defense Advanced Research Projects Agency), that has the title “Safe Genes,” that’s about safe ways of manipulating genes and genomes. And one of the strategies that groups are using to do that is using these anti-CRISPRs.
Do you think that CRISPR helps us get closer to understanding how all the pieces in cells are working together rather than just separately?
I think it will increasingly play that kind of a role in the future.
Let’s go back to neuroscience, because there’s a case where CRISPR has come to the fore in studies of the development of the brain. Researchers haven’t been able to figure out how many different types of cells are in the brain. We also don’t know how the brain develops in the sense of its 3D architecture. If you start with a stem cell or a few stem cells, how does that develop into an entire brain, and what’s the map of the brain?
There’s a lot of interest right now in using CRISPR to do what’s called lineage mapping. If you have a population of cells that develop from a single cell or a small collection of cells, you can track how cells from that starting population give rise to their progeny by introducing a little edit to their DNA to mark them.
Several research teams are using CRISPR this way to figure out where these daughter cells end up in the brain and even what kinds of cells they become. I think these kinds of experiments will lead to a more fundamental understanding of tissue development — in particular, in the brain — that hasn’t been possible before.
That does sound promising.
I’ll give you another example. There are interesting cases — and we’re finding more and more of these as people get their DNA sequenced — of families in which everybody has a certain allele, a certain DNA sequence of a gene, but only some of them have a disease that’s associated with that allele. The others don’t. So you know that there’s something in the DNA of the people who are unaffected that suppresses a negative impact of that gene and makes them not susceptible to cancer or whatever other disease they would otherwise succumb to. What are those suppressors?
I think understanding those kinds of genetic interactions is going to be incredibly powerful going forward. Up until now, we haven’t really had a way to do it because, first of all, people weren’t widely going around sequencing their genomes. That’s starting to happen more and more, with companies that offer this and the cost coming down. Then there’s also having a technology that allows genetic manipulation of patient-derived cells. So if you have somebody that comes into a clinic and they have a disease that gets diagnosed, you can take cells from that person and you can cultivate them in the lab. That’s been possible for a while, but what wasn’t possible previously was to do genetics on those cells. Now we can, in living cells that relate to an actual patient.
That sounds like an unexpected benefit of the sequencing technology.
I always like to point out that there’s a certain serendipity to science. It’s wonderful, but it also means that you can’t predict outcomes. CRISPR technology is a great example of that. If you had told me 10 years ago that bacteria had evolved proteins that could be programmed to find and cut any DNA sequence, I would have just laughed. I would have been like, “Yeah, that’s definitely science fiction.”
I think it’s important for people to appreciate that this is how a lot of science happens.
Individuals carrying these ancient ancestors’ DNA are more likely to have slightly elongated, rather than rounded, brains
The researchers are quick to point out that their findings don’t suggest a link between brain size or shape and behavior, but instead offer an exploration of the genetic evolution of modern brains (Philipp Gunz)
Neanderthals may have gone extinct some 40,000 years ago, but thanks to long-ago species interbreeding, their genes live on in modern humans.
The implications of this genetic inheritance remain largely unclear, although previous studies have proposed links with disease immunity, hair color and even sleeping patterns. Now, Carl Zimmer reports for The New York Times, a study recently published in Current Biology offers yet another example of Neanderthals’ influence on Homo sapiens: Compared to individuals lacking Neanderthal DNA, carriers are more likely to have slightly elongated, rather than rounded, brains.
This tendency makes sense given Neanderthals’ distinctive elongated skull shape, which Science magazine’s Ann Gibbons likens to a football, as opposed to modern humans’ more basketball-shaped skulls. It would be logical to assume this stretched out shape reflects similarly protracted brains, but as lead author Philipp Gunz of Germany’s Max Planck Institute for Evolutionary Anthropology tells Live Science’s Charles Q. Choi, brain tissue doesn’t fossilize, making it difficult to pinpoint the “underlying biology” of Neanderthal skulls.
To overcome this obstacle, Gunz and his colleagues used computed tomography (CT) scanning to generate imprints of seven Neanderthal and 19 modern human skulls’ interior braincases. Based on this data, the team established a “globularity index” capable of measuring how globular (rounded) or elongated the brain is. Next, Dyani Lewis writes for Cosmos, the researchers applied this measure to magnetic resonance imaging (MRI) scans of around 4,500 contemporary humans of European ancestry, and then compared these figures to genomic data cataloguing participants’ share of Neanderthal DNA fragments.
Two specific genes emerged in correlation with slightly less globular heads, according to The New York Times’ Zimmer: UBR4, which is linked to the generation of neurons, and PHLPP1, which controls the production of a neuron-insulating sleeve called myelin. Both UBR4 and PHLPP1 affect significant regions of the brain, including the part of the forebrain called the putamen, which forms part of the basal ganglia, and the cerebellum. As Sarah Sloat explains forInverse, the basal ganglia influences cognitive functions such as skill learning, fine motor control and planning, while the cerebellum assists in language processing, motor movement and working memory.
In modern human brains, PHLPP1 likely produces extra myelin in the cerebellum; UBR4 may make neurons grow faster in the putamen. Comparatively, Science’s Gibbons notes, Neanderthal variants may lower UBR4 expression in the basal ganglia and reduce the myelination of axions in the cerebellum—phenomena that could contribute to small differences in neural connectivity and the cerebellum’s regulation of motor skills and speech, the study’s lead author Simon Fisher of the Netherlands’ Max Planck Institute for Psycholinguistics tells Gibbons .
Still, the effects of such gene variations are probably negligible in living humans, merely adding a slight, barely discernible elongation to the skull.
“Brain shape differences are one of the key distinctions between ourselves and Neanderthals,” Darren Curnoe, a paleoanthropologist from Australia’s University of New South Wales who was not involved in the study, tells Cosmos, “and very likely underpins some of the major behavioural differences between our species.”
In an interview with The New York Times, Fisher adds that the evolution of UBR4 and PHLPP1 genes could reflect modern humans’ development of sophisticated language, tool-making and similarly advanced behaviors.
But, Gunz is quick to point out, the researchers are not issuing a decisive statement on the genes controlling brain shape, nor the effects of such genes on modern humans carrying fragments of Neanderthal DNA: “I don’t want to sound like I’m promoting some new kind of phrenology,” he tells Cosmos. “We’re not trying to argue that brain shape is under any direct selection, and brain shape is directly related to behaviour at all.”
How many years did you spend working on creating GM potatoes? Was this all lab-based work or did you get out to see the farms that were growing the potatoes?
During my 26 years as a genetic engineer, I created hundreds of thousands of different GM potatoes at a direct cost of about $50 million. I started my work at universities in Amsterdam and Berkeley, continued at Monsanto, and then worked for many years at J. R. Simplot Company, which is one of the largest potato processors in the world.
I had my potatoes tested in greenhouses or the field, but I rarely left the laboratory to visit the farms or experimental stations. Indeed, I believed that my theoretical knowledge about potatoes was sufficient to improve potatoes. This was one of my biggest mistakes.
Have the GM potatoes you helped create been approved by the FDA and EPA in the U.S. or indeed elsewhere in the world?
It is amazing that the USDA and FDA approved the GM potatoes by only evaluating our own data. How can the regulatory agencies assume there is no bias? When I was at J.R. Simplot, I truly believed that my GM potatoes were perfect, just like a parent believes his or her children are perfect.
I was biased and all genetic engineers are biased. It is not just an emotional bias. We need the GM crops to be approved. There is a tremendous amount of pressure to succeed, to justify our existence by developing modifications that create hundreds of millions of dollars in value. We test our GM crops to confirm their safety, not to question their safety.
The regulatory petitions for deregulation are full with meaningless data but hardly include any attempts to reveal the unintended effects. For instance, the petitions describe the insertion site of the transgene, but they don’t mention the numerous random mutations that occurred during the tissue culture manipulations. And the petitions provide data on compounds that are safe and don’t matter, such as the regular amino acids and sugars, but hardly give any measurements on the levels of potential toxins or allergens.
The Canadian and Japanese agencies approved our GMO potatoes as well, and approvals are currently considered in China, South Korea, Taiwan, Malaysia, Singapore, Mexico, and the Philippines.
What was your role at Monsanto and J.R. Simplot?
I led a small team of 15 scientists at Monsanto, and I directed the entire biotech R&D effort at Simplot (up to 50 scientists). My initial focus was on disease control but I eventually considered all traits with commercial value. I published hundreds of patents and scientific studies on the various aspects of my work.
Why did you leave firstly Monsanto and then later J.R. Simplot?
I left Monsanto to start an independent biotech program at J.R. Simplot, and I left J.R. Simplot when my ‘pro-biotech’ filter was wearing thin and began to shatter; when I discovered the first mistakes. These first mistakes were minor but made me feel uncomfortable. I realized there had to be bigger mistakes still hidden from my view.
Why have you decided to reveal information about the failings of GM potatoes after spending many years creating them?
I dedicated many years of my life to the creation of GMO potatoes, and I initially believed that my potatoes were perfect but then I began to doubt. It again took me many years to take a step back from my work, reconsider it, and discover the mistakes. Looking back at myself and my colleagues, I believe now that we were all brainwashed; that we all brainwashed ourselves.
We believed that the essence of life was a dead molecule, DNA, and that we could improve life by changing this molecule in the lab. We also assumed that theoretical knowledge was all we needed to succeed, and that a single genetic change would always have one intentional effect only.
We were supposed to understand DNA and to make valuable modifications, but the fact of the matter was that we knew as little about DNA as the average American knows about the Sanskrit version of the Bhagavad Gita. We just knew enough to be dangerous, especially when combined with our bias and narrowmindedness.
We focused on short-term benefits (in the laboratory) without considering the long-term deficits (in the field). It was the same kind of thinking that produced DDT, PCBs, Agent Orange, recombinant bovine growth hormone, and so on. I believe that it is important for people to understand how little genetic engineers know, how biased they are, and how wrong they can be. My story is just an example.
A Northwestern University team spent about eight years meticulously studying the human genome and all of its various chemicals and processes it uses to regulate itself, and it has discovered what it bills as a seemingly foolproof “self-destruct pathway,” that could be utilized for healing, to destroy any type of cancer cell one can think of.
The mechanism they found involves the creation of things called siRNAs, small RNA molecules that serve to interfere with a multitude of genes that are essential to the destructive proliferation of malignant, fast-growing cells. It is said that these siRNAs reportedly have little effect on our healthy, good cells. However, one might see already that if this cancer fighting strategy has risks, people should take a very close look at them.
They say insight was provided by two recent studies, and Marcus Peter, the research leader along with his colleagues have outlined in detail the series of events that the siRNA molecules trigger in our bodies.
They called this process of siRNA molecules causing cancer cell death DISE, or Death By Induced Survival gene Elmination. The team managed to identify six-nucleotide-long sequences that would be required for inducing this state of “DISE.”
It was explained that when researchers examined the nucleotide sequences of the various noncoding, non-protein translating RNA molecules our bodies produce naturally to selectively inhibit the expression of genes, they made the discovery that DISE-associated sequences are there, present at one end of several tumor suppressing RNA strands.
Yet another investigation concluded that those sequences can also be found throughout the genome, embedded in protein-coding sequences.
“We think this is how multicellular organisms eliminated cancer before the development of the adaptive immune system, which is about 500 million years old,” Peter said last year, in a statement. “It could be a fail-safe that forces rogue cells to commit suicide. We believe it is active in every cell protecting us from cancer.”
However, there was one small problem: they still had to determine just how the body produces these free siRNAs that are capable of producing DISE. That’s how complicated the body gets.
In another new study, a breakthrough came as it was published last month in eLife, and in that one Peter and his team observed the body making these molecules, as our cells basically chop a larger strand of RNA, that codes for a cell death cycle protein known as CD95L, into multiple siRNAs.
A series of experiments were conducted, and then they managed to show that the exact same cellular machinery could be utilized in converting other large protein-coding RNAs into these DISE siRNAs.
Even more remarkably, they found that somewhere around three percent of all the coding RNAs in our entire genome could be “processed” to serve the purpose.
“Now that we know the kill code, we can trigger the mechanism without having to use chemotherapy and without messing with the genome,” Peter said last month in a press conference.
Now, they want to make “next-generation medications” and “gene therapy,” and while this sounds like it makes a lot of sense, it’s true that people would be wise to keep their wits about them and know just what they are signing up for if they proceed with some new treatment resulting from this research.