“We aren’t going to get sick, are we?” my roommate Brett asked me. He cringed as I knelt down and stuffed a plate of E. coli bacteria—which came as part of the DIY CRISPR–Cas9 kit I bought online—into our fridge next to cartons of eggs, strawberry jam, bottles of beer and a block of cheese.
“No, we won’t. The label says ‘non-pathogenic,’” I replied, trying to sound assuring. But honestly, I had no clue what I was doing. I nudged all the food up against the fridge wall, and left a two-inch border around the plate of living cells—a no man’s land between the microbes and our dinner. A couple inches probably would not stop the bugs, but I figured it couldn’t hurt.
CRISPR–Cas9 (or CRISPR, for short) has given scientists a powerful way to make precise changes to DNA—in microbes, plants, mice, dogs and even in human cells. The technique may help researchers engineer drought-resistance crops, develop better drugs, cure genetic disorders, eradicate infectious diseases and much more. Ask any biologist, and they’ll likely tell you that CRISPR is revolutionary. It’s cheap and effective, and in many cases, it works much better than older methods for making genetic modifications. Biologists will also tell you that CRISPR is very easy to use. But what does “easy to use” mean?
I am not a DIY scientist, much less a professional scientist. You won’t find me swabbing my cheek cells for DNA or tinkering with yeast in a lab on the weekend. But I wondered: Is CRISPR so easy that even amateurs like me can make meaningful contributions to science? And also, does this new technique make gene editing so accessible that we need to worry about DIY scientists cooking up pandemic viruses in their basements? If you Google ‘DIY CRISPR,’ stories such as “What Happens If Someone Uses this DIY Gene Hacking Kit to Make Mutant Bacteria?” pop up.
I attempted to find answers to all these questions myself, starting with the plate of bacteria in the kitchen of my San Francisco apartment.
CUT AND SPLICE
CRISPR stands for “clustered regularly interspaced short palindromic repeats.” The CRISPR system is made up of two components: a protein called Cas9 and a guide RNA, a string of nucleic acid molecules with a certain genetic code. Put them together, and they create a tool you can use to tweak an organism’s genome. To do this, CRISPR searches the organism’s DNA for a certain sequence—specifically, the one encoded by the guide RNA, which holds the inverse sequence of your target DNA. “Cas9 opens up the DNA, it separates the strands of the double helix in a very small area, and allows the guide RNA to pair with one of the strands,” explains Dana Carroll, a professor of biochemistry at the University of Utah. “If it is a good match, cutting occurs. If it is not a good match, Cas9 and [the] guide RNA fall off and try again somewhere else.” When it finds the right sequence, the Cas9 protein slices the DNA at that precise spot.
At this point, if you leave the cell alone, it will usually mend CRISPR’s cut—but it will occasionally also make a mistake in the repair process, breaking a gene or other parts of the genome. Since CRISPR repeatedly goes back and slices the DNA again after the cell mends it, the gene eventually breaks, or, in technical terms, gets knocked out. And, if you add new DNA, the cell may incorporate it while fixing the cut. This means you can insert DNA where you want to in the genome—you just need to know the organism’s genetic sequence of your desired target area.
Scientists originally discovered this sophisticated system in archaea and bacteria, which deploy CRISPR to chop up invading viruses. But a few years ago, researchers figured out how to repurpose CRISPR to run in pretty much any living thing they want. And now it’s made genetic engineering easier than ever before.
For my own experiment, everything I needed came in a small cardboard box—an assortment of bottles, tubes, plates, powders and liquids (plus E. coli). I ordered my kit for $130 from the crowd-funding site Indiegogo as part of a campaign created by Bay Area biohacker Josiah Zayner. Zayner has a PhD in molecular biophysics and spent two years as a research fellow at NASA. He ran the crowdfunding campaign out of his apartment, and by the end of it, he had raised over $70,000 and sold 250 DIY CRISPR kits—one of which now sat on my kitchen table. Zayner has now sold over a thousand kits, largely on his company’s website, The Odin.
The idea behind the kit experiment is quite simple. The goal: modify the E. coli so that it can grow on an antibiotic called streptomycin, which normally kills bacteria. With materials and instructions from the kit, I will introduce CRISPR into the bacteria cells, and use it to rewrite a tiny part of their DNA, creating genetically altered cells that happily thrive on streptomycin. In the end, CRISPR will track down and then change only a single base pair (which are the building blocks for DNA) out of the 4.6 million base pairs in the E. coli genome. It will swap out the chemical compound adenine for cytosine—or, in terms of the genetic alphabet, an “A” for a “C.” Because of that tiny code change, my bacteria cells will make the amino acid lysine instead of another one, threonine. If my gene editing succeeds, this will stop streptomycin from interfering with the E. coli.
On a Monday afternoon in May, I donned latex gloves and spread newspaper over the dinner table. I grabbed three small plastic tubes from the freezer, picked up a pipette—a hand-held instrument, used in labs to measure out liquids—from my DIY kit, and started adding the CRISPR ingredients to tubes of E. coli.
I put in the Cas9 protein and guide RNA, which came as liquids in small, plastic test tubes, to my bacteria cells. Then I dipped my pipette into a small tube of DNA and tried to suck up 10 microliters. Nothing came out. I squinted at the tiny drop of clear liquid, and realized it had frozen solid. I had no clue why. Uh oh, I hope that’s not a problem. Would it harm my experiment? I had absolutely no idea, so I just waited for the DNA solution to thaw and then squirted it into the bacteria tube. After several more steps, I spread my CRISPR’d bacteria onto three plastic plates and put them in my laundry room. The instructions said to wait 24–48 hours, then check for small white dots of bacteria. If I saw dots, CRISPR had done its job of splicing in the streptomycin-resistance gene. If not … well, failure is also part of the scientific process.
I had no problem conducting the experiment—CRISPR is easy, I concluded. I basically just measured, scraped and stirred a bunch of ingredients, occasionally cooling them or heating them up. But for all the godlike powers that I imagined CRISPR gave me, I actually had little say over what I did to my bacteria. Everything was predetermined, with instructions laid out for me like steps in a cookbook: “Add 100 microliters Transformation mix to a new centrifuge tube,” “Incubate this tube in the fridge for 30 minutes,” and so on. Ultimately, I had made zero decisions. Of course, I could have designed a custom-made CRISPR experiment—but it would have taken more time, more materials, more money, and a lot more knowledge than I currently had.
I inspected my bacteria 48 hours later. Crossing my fingers, I lifted the lid on the first plate. No white dots. Then the second plate: nothing. My stomach sank with disappointment. Then I gently raised the lid off the third plate, and saw … something. The plate had two faint milky white circles. Had CRISPR worked? Maybe. But then why did one plate have white spots, but not the other plates? I’d followed the same steps for each of them. Maybe my mind was playing tricks on me. Or perhaps I had contaminated the third plate. If only I could show my plates to someone who knew how to interpret them. Like a scientist. Unfortunately, there was no one to ask in my kitchen.
OPEN TO EVERYONE
A few weeks later, I drove 40 miles south of San Francisco to meet a DIY scientist named Johan Sosa, who knows way more about CRISPR than I do. We met at BioCurious, a community laboratory in Sunnyvale where he works most weekends and some evenings. Located in Santa Clara, BioCurious is a co-working space outfitted with science equipment and shared by “scientists, technologists, entrepreneurs, and amateurs who believe that innovations in biology should be accessible, affordable, and open to everyone,” according to its Web site. The lab is funded by donations and members—Sosa is one of its several dozen members.
At six foot, five inches, Sosa towers over most people. “I’m probably the tallest DIY biologist,” he jokes. He laughs easily, which offsets his height to lend him a gentle, laid-back manner. He is 40 years old, his dark hair flecked with silver. Originally from Sri Lanka, Sosa came to the U.S. for college at 15 years old to study computer science, and he’s worked for Bank of America and IBM as a computer security specialist and software engineer. He now has a day job in computer security—but he spends most of his free time at BioCurious. “You could say I have no life,” he chuckles, “This is my biggest hobby.” He has learned everything he knows about science (both theory and lab techniques) from others at BioCurious, by reading science papers, watching YouTube videos, attending lectures, and also through trial-and-error in his own research.
Sosa is one of a few DIY scientists at BioCurious using CRISPR. He first read about the technique in 2012, from one of biochemist Jennifer Doudna’s papers in the journal Science. Doudna, a professor at University of California, Berkeley, is one of the pioneers of CRISPR. “I didn’t think it was that big of a deal, because I knew there were already other ways of modifying DNA,” he recalls, “But I did think, ‘This is something I could do.’” He started experimenting with CRISPR in 2013.
On a muggy, overcast afternoon, I followed Sosa inside BioCurious. We strolled through a lobby and into a large windowless room. It had big cabinets crammed with bottles of liquid, racks of latex gloves, a giant bio-hood, microwave and fridge. Microscopes, scales, centrifuges and a bunch of other well-worn science equipment lay scattered across lab desks. A calm thrum of buzzing machinery filled the room, and test tubes shook quietly in an incubator nearby. Johan walked around the room, searching for a thermometer. “One thing about a DIY lab is that you leave something somewhere, and it always ends up somewhere else,” he told me.
I had joined Sosa at BioCurious so I could learn more about what CRISPR means for DIY scientists, and also to do a (hopefully) more successful experiment, with his help. We decided on a very basic goal: we would use the powerful editing tool to cut DNA he had already extracted from yeast cells. This task is easier than the one I tried in my kitchen, because you don’t need to get CRISPR inside cells in order to slice the DNA. Professional scientists might use such a method as an intermediate step, such as when they need to cut and paste DNA together to make a gene as part of a bigger research project. “That’s a type of experiment everyone does every day,” explains Charles Gersbach, a professor of biomedical engineering at Duke University—though he notes that traditionally, researchers have used a type of protein called a restriction enzyme, not CRISPR, to do this.
Sosa and I pulled on latex gloves and carefully pipetted liquids into tubes to make our guide RNA from scratch—we first synthesized DNA strands with a specific sequence we wanted, used that as a template for the RNA, then destroyed the DNA and isolated the guide RNA from our test tube mixture. Later, we put the RNA in a new test tube, along with the other materials needed to make CRISPR work in this experiment: protein buffer, bovine serum albumin (a protein isolated from cows), water. Sosa sucked up the yeast DNA in his pipette. Without warning, the plastic needle-nose tip fell off into the tube of DNA. Someone, he told me, had donated the tips to their lab, but they weren’t the correct size. “I guess you got the full DIY experience,” he smiled, and pushed the plastic tip back onto the pipette. Then he picked up the Cas9 protein. “Here’s the world-famous Cas9,” he said, and handed it to me. I added it to our test tube.
Until CRISPR, DIY scientists didn’t have an easy, cheap or reliable way to precisely edit DNA. Many of them couldn’t afford the pricey and imperfect tools that professional scientists used for gene editing at the time. “Before CRISPR, there was TALENS [transcription activator-like effector nucleases] and zinc finger nucleases—older technologies that were not as precise or reliable,” explains Sosa. “They were out of the budget and the time constraints of DIY scientists.” Sosa says that if a DIY-er used those other technologies, it might cost him or her thousands of dollars to do a genetic engineering experiment. But with CRISPR, it’s vastly more affordable, especially if you want to attempt an experiment more than once. “With TALENS, you try it once and fail,” says Sosa. “With CRISPR, you can try it multiple times. That alone is a big deal.”
This means that CRISPR gives DIY-ers a whole new way to do science. So far, Sosa and his lab mates have tried out CRISPR in a number of ways: cutting yeast genomes, slicing DNA inside E. coli cells, and attempting to modify the CRISPR system by shrinking it or attaching other molecules to it. Sosa has goals for his CRISPR research. “I want to understand how a cell really functions, and what are all the little things that happen in it,” he explains. “And when something goes wrong [such as in diseases], how to fix it or make it do what I want.”
After several hours, Sosa and I checked to see if CRISPR had cut our yeast DNA. We dyed our DNA-CRISPR mixture blue and ran it through an electrically charged gel, which separates bigger DNA pieces from smaller ones. Tiny channels in the gel run from one charged end to the other, and the sliced DNA strands are pulled through them towards the positively charged side. If our experiment succeeded, we should see two blue bands for the short CRISPR-cut DNA strands in one spot, and one blue band for a longer, uncut piece of DNA (our control) in another location.
Sosa carried the gel into the bathroom, where we turned off the lights and looked at it under blue light. I held my breath while I inspected the gel for markings. One light-blue band gleamed in the dark—the control—and another single band lit up the spot where we should have seen our CRISPR’d DNA. “I don’t know what happened, but it doesn’t look right,” Sosa said, “I don’t think it worked.”
I left the lab feeling defeated, and headed back to San Francisco. Sosa texted me a few minutes later.
Hey, I figured out what happened. There was no DNA to start with, he wrote.
What happened? I texted back.
I think the DNA had either degraded or gotten too diluted, he wrote.
Even if we had got all the other parts (RNA, proteins, etc.) working, it didn’t matter. We hadn’t given CRISPR any DNA to cut. My second attempt at CRISPR had utterly failed.
BETTER, FASTER, CHEAPER
My own frustrating struggles with CRISPR aside, I wanted to see what professional biologists are doing with CRISPR, so I visited the lab of Nipam Patel at the University of California, Berkeley. After a quick tour of the lab, I sat down and stared into the microscope at a small, writhing marine creature: Parhyale hawaiensis, commonly called a beach hopper. At one centimeter long, Parhyale looks puny—you’d step on it at the beach without even noticing. But under the microscope, this female hopper resembled a giant translucent shrimp with many powerful, kicking legs. Parhyale is the star of this lab. “We’re looking at how you develop an individual body,” explains Erin Jarvis, a PhD student in Patel’s lab, “And also how you build a body form over evolutionary time.” And they’re using CRISPR to do it.
With CRISPR, these researchers knock out so-called Hox genes in Parhyale. Hox genes are found in all animals, including humans, and they control the development of their body plans. Among other things, they determine what appendages—such as swimming legs, claws and antennae—grown on which section of the body. Knock out a certain Hox gene with CRISPR and Parhyale will grow forward-walking legs where it should have jumping legs, for example.
Parhyale has nine Hox genes, and Patel’s team has knocked out seven of them. The researchers also have plans to add completely new genes to Parhyale using CRISPR—they’ve already done it once, by inserting a gene that codes for green fluorescent proteins, which allowed the researchers to visualize where a specific Hox gene is expressed in Parhyale. “From an evolutionary perspective, this [gives] us insight into how body plans evolve between species,” when comparing Parhyale to, for example, the well-studied fruit fly, Drosophila, explains Patel. “We believe that such evolutionary patterns help us understand the general mechanisms by which evolution creates animal diversity.… What we learn improves our knowledge about the function of these genes in other animals, including humans.”
CRISPR has transformed how Patel and his colleagues do their research. His lab has looked at Parhyale for about 20 years now. Before CRISPR they used another technique to knock out genes that required a lot more money, and even then, it wasn’t very efficient. It cost them about $900 to knock out a single gene in a group of Parhyale embryos with their other method. The technique, called “RNA interference,” silences expression of a gene—it doesn’t genetically knock it out as CRISPR does. The problem was, sometimes the method didn’t work at all.
Now it costs them less than $100 to knock out a gene. “Suddenly, with CRISPR, you don’t have to decide, ‘Which one gene do I want to put all my resources into?’” Jarvis says, “You can try a lot of different genes.” And with CRISPR, they’re able to break genes in up to 75 percent of Parhyale embryos, versus a maximum 25 percent success rate with the old technique. Even better, they now have the ability to mutate several genes at once with CRISPR, which means they can now see how genes interact. When the researchers had tried to mutate multiple genes with their old technique, it rarely worked. (Though Patel notes that the older RNA technique is still very useful for certain applications).
Their research takes less time with CRISPR, too—in a study Patel’s lab published in Current Biology in 2015, they knocked out six Hox genes in about a year. Before that, they had already spent years trying to break a specific Hox gene with their old method, but were never able to do it. “Everything just goes faster,” says Patel, a professor of genetics, genomics and development. “CRISPR-Cas9 is an incredibly elegant system, and it’s very easy to control.” It also makes it simpler to study more exotic creatures (beyond the standard flies and mice), such as animals like Parhyale or butterflies. “It’s always been hard to work with a new organism,” says Jarvis, “CRISPR is awesome because suddenly, you don’t have to spend decades developing a model.” As long as you have the sequence of the gene you want to target, you’re set.
Patel’s lab is hardly the only one capitalizing on CRISPR—scientists around the world are exploring all sorts of different uses for the gene editing tool, like wiping out malaria-spreading mosquitoes, finding new ways to treat cancer, or engineering disease-resistant crops. In July, researchers announced they had successfully edited the genome of viable human embryos with CRISPR; the technique allowed them to fix a disease-causing mutation in the embryos’ DNA (though some are now skeptical of the researchers’ results). Just a few weeks later, scientists in Massachusetts reported they had made a significant advance towards pig-to-human organ transplants. They used CRISPR to inactivate 25 viruses intrinsic to pigs’ genomes, overcoming a big obstacle in making porcine transplants safe for humans.
I had reached the end of my CRISPR experiment—so what had I learned? First, I found out it was not completely crazy for my roommate to wonder whether a DIY CRISPR kit in our fridge would make us sick. This year, German authorities restricted imports of the Odin DIY CRISPR bacteria kit after the Bavarian Health and Food Safety Authority tested two kits and found them to contain potentially pathogenic bacteria. But even the European Center for Disease Prevention and Control concluded that there was little to worry about—that “the risk of infection by the contaminating strains in the kit is low for the users … assuming that they are healthy people.” (Zayner declined to comment on the record about the incident, but he publicly posted a response on Twitter, where he criticized the methodology used by the Bavarian agency and denied wrongdoing by his company. The kits are still available for purchase online through The Odin.)
As for my bigger question—could untrained DIY-ers actually achieve scientific breakthroughs?—I asked academic researchers what they thought. Dana Carroll, for his part, believes amateurs could make meaningful discoveries. “In the professional science community, people keep coming up with new ways to use this technology—people are really only limited by their imagination,” he explains. “It’s possible that people working in their garages or their kitchens will come up with a novel application or a solution to a problem that professionals just haven’t gotten around to.” And Carroll says it would be easy for a DIY-er to share any discoveries with researchers, by attending their talks or simply by contacting them through their Web sites. Yet he notes that the DIY community faces limitations, because amateur scientists likely would lack the necessary resources. “It’s unlikely they will bring a major application all the way to fruition,” he says, “But they could certainly get started on something.”
Finally, what about the nightmare scenario: Is CRISPR so easy to use that we need to worry about biohackers—either accidentally or intentionally—creating dangerous pathogens? Carroll and others think that the danger of putting CRISPR in the hands of the average person is relatively low. “People have imagined scenarios where scientists could use CRISPR to generate a virulent pathogen, ” he says. “How big is the risk? It’s not zero, but it’s fairly small.” Gersbach agrees. “Right now, it’s difficult to imagine how it’d be dangerous in a real way,” he explains, “If you want to do harm, there are much easier and simpler ways than using this highly sophisticated genetic editing technique.”
Back in Patel’s lab, Jarvis replaced the squirming female beach hopper under my microscope with a tiny Parhyale embryo. Jarvis told me she knocked out a Hox gene called Abd-B in this one—the embryo will grow jumping legs where it should have swimming legs, and forward walking legs instead of anchor legs. At this point, it just looked like an opaque ball of goo to me.
Next to me, another grad student examined a fragment of a brown and gold butterfly wing—Patel’s lab is also knocking out butterfly genes with CRISPR to see how they build wing color. “An old grad student used to joke that we were genetically modifying the wings to make the Mona Lisa,” Jarvis told me. I laughed and glanced back under the microscope. A puff of my breath suddenly struck the Parhyale embryo. It danced wildly around the petri dish, like a grain of sand caught in a windstorm.