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My Genetk Design Kits arrived in a box bearing a stylized version of Yggdrasil, the world tree in Norse mythology, with a twist of the DNA double-helix as part of its trunk. On the sides of the box, Odin's ravens, Huginn (thought) and Muninn (memory), exchange a strand of DNA. Odin had, in fact, sent me the box, and by Odin, I mean The ODIN—The Open Discovery Institute, a company that aims to make do-it-yourself genome editing easy. I was ready to start genetically editing bacteria at home.
This is possible because of CRISPR, a technology that is already revolutionizing food, medicine, and more. CRISPR comprises two key molecules. One is the Cas9 protein, an enzyme that can cut two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed. The second is a single-strand RNA that can identify and guide the protein to exactly the site in a genome that a researcher wants to engineer. The system has been likened to precise molecular scissors.
Using the handy tools sent in the kit, I was set to re-engineer some nonpathogenic E. coli in my kitchen. That might sound terrifying; surely journalists shouldn't be trusted to build superbugs. Relax. The lab-created strain provided in the kit was developed to be easy to engineer and does not live in the wild. While CRISPR holds incredible potential for in-lab and at-home genetic modification and experimentation, my efforts were strictly school science fair stuff—my modified bacteria posed no civilizational risk, and the process of creating them was fun, fascinating, and empowering.
The CRISPR revolution began in 2012, when Jennifer Doudna of Berkeley and Emmanuelle Charpentier of Sweden's Umeå University published an article in Science describing how elements of a bacterial immune system could be used as a very precise gene-editing tool. In 2013, Broad Institute researcher Feng Zhang showed that CRISPR could edit genes in human cells. (A big CRISPR patent fight between Berkeley and the Broad Institute is now underway.)
Since then, there's been a flood of research into therapeutic uses of the technique. Last year, Shoukhrat Mitalipov of Oregon Health and Science University used CRISPR to correct a genetic mutation in human embryos that causes heart disease. Other researchers are working on CRISPR therapies to cure Huntington's, Parkinson's, sickle cell anemia, Duchenne muscular dystrophy, and various congenital blindnesses. Chinese physicians are already running trials in which they use CRISPR to rev up cancer patients' immune cells. This summer a trial at the University of Pennsylvania will try to use CRISPR techniques to treat multiple myeloma, sarcoma, and melanoma. Some researchers think a one-time CRISPR "vaccination" could edit a specific gene associated with cholesterol, thus lowering a patient's risk of various cardiovascular diseases.
The technology will also radically change how we grow our crops, make our foods, and curate our natural environment. With gene editing, researchers can make changes to a plant or animal's existing genome—a departure from the conventional genetic modification technique, which inserts useful genes taken from other creatures. As a result, many researchers and developers argue that genome editing should be much more lightly regulated than conventional genetic engineering has been.
The ODIN already sells a kit allowing home gene jockeys to brew green glowing beer. (The kit enables a user to inject a gene for a harmless green fluorescent protein derived from jellyfish into the yeast.) Researchers at the University of California have used CRISPR to edit flavor directly into yeast, so brewers no longer have to add finicky and expensive hops to make the IPAs I relish.
Plant breeders are using CRISPR to improve various crops. DuPont has a CRISPR-edited waxy corn that is resistant to drought and disease. Wang Wei of Kansas State University has edited 25 wheat genes to dramatically increase yields. A Spanish research group has edited out the wheat genes that produce the gluten proteins that bedevil folks with celiac disease. Researchers in Colombia are CRISPRing rice and cassava to make them resistant to diseases, and altering beans to make them more easily digestible.
Animal breeders have deployed CRISPR to eliminate dangerous horns on dairy cattle and to skew the production of calves toward males in order to boost beef production. The ODIN's founder, the biohacker Josiah Zayner, injected himself last October with CRISPRed DNA designed to silence the myostatin genes that regulate muscle growth. The goal was to enhance his physique by letting his muscles get larger than they otherwise would. So far he has reported no results from the experiment. But whether or not Zayner manages to use CRISPR to knock out his own myostatin genes, the technique has been used successfully to make more ham by generating extra-muscular pigs.
The technology can also be used to create "gene drives." A gene drive works by making sure that all copies of the natural gene are replaced with the engineered versions in the progeny of CRISPRed organisms. This causes a desired trait to spread rapidly through a whole population in a natural environment. It would be possible, for example, to edit resistance to the malaria parasite or the Zika virus into entire populations of mosquitoes. A gene drive could also be constructed such that only males of an undesired species are born.
In 2015, Science hailed CRISPR gene editing as the breakthrough of the year. It is, the magazine declared, "only slightly hyperbolic to say that if scientists can dream of a genetic manipulation, CRISPR can now make it happen." As you can see from my very incomplete review of the rapid progress being made, it is hardly hyperbolic at all.
With great power comes great responsibility, of course, and the fight over the regulation of in-lab and at-home genetic modification is raging. You may want to order a CRISPR kit soon, in case the prohibitionists win.
With my wife's tolerance, I stored my kit in our refrigerator and set up a gene-editing laboratory on a red towel on our kitchen counter. Thankfully, our dinner guests were too polite to mention the petri dishes streaked with bacteria or the other lab equipment spread out in the kitchen.
For those of us who are not practiced lab jockeys, the instruction booklet that accompanies the kit is a bit opaque. Fortunately, there are some online videos to show novices how to brew up agar and to pipette biochemicals into microcentrifuge tubes.
Besides the various mixing bottles and measuring tubes, the Genetk Design Kits box came with nitrile gloves, LB Agar powder on which to grow bacteria, and LB Agar powder spiked with the antibiotics streptomycin and kanamycin. Containers held the nonpathogenic E. coli, a solution of calcium chloride and polyethylene glycol (the "bacterial transformation buffer"), Cas9 plasmid, guide RNA, and template DNA for antibiotic resistance.
With help from those online videos, I made both regular and antibiotic-spiked agar and poured each mixture onto seven petri dishes, where the agar congealed. Next, I used a plastic inoculation loop to scoop some bacteria out of their bottle and streak them onto a couple of the agar dishes to grow. As a control, I also spread some onto the plates spiked with antibiotics to see if the drugs would prevent bacterial growth.
After incubating at room temperature for about 24 hours, the bacterial streaks on the regular agar plates turned white and widened. Due no doubt to my sloppy lab work and the ubiquitous presence of bacteria, several of the regular agar plates I did not streak grew nice round wild colonies as well. Nothing was seen growing on the streptomycin/kanamycin plates.
The next day, I scraped fresh bacteria off of the plates with another inoculation loop and dumped these into the tube containing the transformation buffer, a substance that basically opens up the bacteria so that the elements of the CRISPR system can sneak in to engineer the target gene.
After refrigerating the bacteria in the transformation buffer for 30 minutes, I heat-shocked them in 108-degree water—measured using a meat thermometer—for 30 seconds. Then I pipetted 500 microliters of regular agar solution into the transformation tube and let it set for four hours at room temperature.
In this way, I made two batches of genetically engineered bacteria. The genomes of E. coli consist of about 4 million DNA base pairs; the goal of this experiment was to change just one of those, which should be enough to allow the bacteria to resist the streptomycin.
What is supposed to happen next is that the Cas9 protein incorporates the guide RNA. The particular guide RNA supplied by The ODIN consists of trans-activating CRISPR RNA (tracrRNA), which binds to the Cas9 protein and links to the CRISPR RNA (crRNA), which in turn targets the DNA in the genome to be edited. In addition to the Cas9 editing system, the bacteria have been flooded with copies of template DNA that differs from the region on the gene targeted for engineering by one base pair.
CRISPR guides the Cas9 complex to the bacteria's rpsL gene, where it makes a cut in both strands of the DNA. When such double strand breaks occur, bacteria have a natural process that seeks to repair them by searching for copies of the broken gene elsewhere in their genomes and then matching the copies.
The rpsL gene is basically a recipe that instructs cellular machinery on how to produce the S12 protein, which is crucial to the operation of ribosomes—the complex macromolecules that make and repair essential proteins in the bacteria, keeping it healthy. Streptomycin works by binding to normal S12 proteins, which disables the ribosomes' vital operation and ultimately kills the bacteria.
The DNA base pairs in the template DNA supplied by The ODIN are identical to those surrounding the cut except for the one base pair that is to be engineered. This tricks the bacteria into using the template to repair the cut made by the Cas9 protein. The only difference is that a guanine/cytosine base pair is substituted for a thymine/adenine base pair. This small change in the rpsL recipe results in a slightly reshaped version of the S12 protein, and that thwarts the antibiotic from binding to and disabling it.
If the transformation is successful, the engineered bacteria will be able to grow despite the presence of streptomycin. The last step, then, was for me to pipette 200 microliters of the (hopefully) transformed bacteria from each of my two batches onto a couple of plates containing drugged agar.
So did it work? After 24 hours, I could detect no obvious growth of bacteria on the antibiotic plates from either of my two initial batches. But most of the bacteria in the 200 microliters taken from the transformation tubes and swabbed onto the petri dish plates will in fact not have been edited. Consequently, the ones that are edited and do survive appear on the plates dosed with antibiotics as small dots, rather than the broad swipes that appear on regular, nondrugged plates.
Fearing that my first two batches had failed, I whipped up a third tube and pipetted some of its bacteria onto a couple of new plates. Hoping that additional time might have worked to transform the bacteria in the first two batches, I pipetted some from those batches onto new plates as well. Finally, as a control, I pipetted bacteria from all three transformed batches onto regular agar plates, where they grew robustly.
More than 60 hours later, by squinting hard, I detected a few tiny scattered colonies on one of the antibiotic-infused plates doused with bacteria from the first transformation batch. As recommended by The ODIN—even though the E. coli I was working with is non-pathogenic—I then sterilized all the plates by dousing them with a bleach solution. While my experiment posed no danger to public health, some worry DIY CRISPRing could create deadly pandemics. But sextillions of daily natural experiments suggest that creating human pathogens is not that easy. Plus, vastly more researchers will be developing beneficial uses of CRISPR, including early warning diagnostics and treatments enabling us to counter any future pandemics.
The experiment was a qualified success at best. Nevertheless, DIY CRISPRing at my kitchen counter reveals just how straightforward and versatile this amazing technology is. Given the spectacular progress researchers are making toward curing diseases, enhancing plants and animals, and curating wild landscapes, it's now clear that this is CRISPR's world; we just have the good fortune to be living in it.
Photo Credit: Ronald Bailey