A Crack In Creation: A Nobel Prize Winner's Insight into the Future of Genetic Engineering

by Jennifer A. Doudna

After all, our work had initially been motivated by curiosity about an entirely unrelated subject: the way that bacteria defend themselves against viral infection. Yet in the course of our research on a bacterial immune system called CRISPR-Cas, we uncovered the workings of an incredible molecular machine that could slice apart viral DNA with exquisite precision.

Homologous recombination occurs most famously during the formation of egg and sperm cells, when the two sets of chromosomes we inherit from our parents are pared down to just one, to be combined with a second set during sexual reproduction. In this process of elimination, cells select a blend of the paternal and maternal chromosomes; each pair of chromosomes engage in their own version of sex, exchanging large chunks of DNA in a way that increases genetic diversity.

elegance,

studies was breathtaking. Within just a few years of my introduction to CRISPR, the field had advanced from a loose collection of interesting but inconclusive studies to a broad, unified theory about the inner workings of a microbial adaptive immune system.

In some respects, the discovery of this part of the bacterial immune system placed bacteria on an equal footing with humans by showing that we both have incredibly complex cellular reactions to infection.

We were discovering this thanks to a massive increase in the number of bacterial and archaeal genomes being sequenced by researchers with easier access to better sequencing tools. CRISPR immune systems were turning out to be highly diverse and could be grouped into multiple different categories, each with its own unique complement of cas genes and Cas proteins.

S. pyogenes and virtually all other members of the Streptococcus genus are known pathogens for a host of mammalian species, including our own. And in humans, a shocking number of illnesses are associated with this particular bacterium. S. pyogenes is one of the top ten causes of deadly infectious diseases for our species, and it’s responsible for over half a million deaths annually. Among the diseases that can be chalked up to S. pyogenes are toxic shock syndrome, scarlet fever, strep throat, and a particularly scary one called necrotizing fasciitis, which has earned S. pyogenes the unpleasant epithet of “flesh-eating bacteria.”

The more I read, the clearer it was that the Cas9 protein was likely to be a key player in the DNA destruction phase of the immune response in Type II CRISPR systems.

Eventually, Krzysztof and Martin performed experiments in which they included not only the CRISPR RNA but also the second type of RNA, called tracrRNA, that Emmanuelle’s lab had found to be required for production of CRISPR RNAs in S. pyogenes. The result was simple but, to us, electrifying: DNA bearing a perfect match to twenty letters in the CRISPR RNA molecule was cleanly cut apart.

In essence, these results simulated what happens in a cell during a CRISPR immune response but with only the minimum of components; no cellular molecules besides Cas9 and the two RNA molecules, which looked similar to the way they’d look inside a Streptococcus pyogenes cell, along with a DNA molecule mimicking the genome of a phage. Of critical importance was the fact that twenty of those DNA letters matched those of the CRISPR RNA, meaning that the CRISPR RNA and one of the two DNA strands should be able to form their own double helix through complementary base pairing. Such an RNA-DNA double helix could be the key to the specificity of Cas9’s DNA-cutting activity.

Academics and physicians alike were hailing CRISPR as the holy grail of gene manipulation: a quick, easy, and accurate way to fix defects in genetic code. In what felt like the blink of an eye, I had been transported from the field of bacteria and CRISPR-Cas biology to the world of human biology and medicine.

Looking at the bottom of the computer screen, I was delighted to see that this was exactly what had transpired: the DNA sequence from the sickle cell patient now looked indistinguishable from the sequence taken from the healthy patient. Using CRISPR, Kiran’s team had perfectly swapped out the disease-causing letter A for the normal letter T without disturbing the genome in any other way. In one simple experiment using a patient’s blood cells, they had shown that the CRISPR-Cas9 system was capable of curing a crippling disease affecting millions of people worldwide.

In November 2013, we founded Editas Medicine with $43 million in financing from three venture capital firms. Just a half a year later, Emmanuelle co-founded another company, CRISPR Therapeutics, with an initial $25 million bankroll, and in November 2014, a third company, Intellia Therapeutics, joined the scene with $15 million in Series A funding. By the end of 2015, these three companies would raise well over half a billion dollars more for research and development of therapies to target numerous disorders, from cystic fibrosis and sickle cell disease to Duchenne muscular dystrophy and a congenital form of blindness, all using the CRISPR technology that Emmanuelle and I had first developed and described.

technology. The days of mentioning CRISPR at a seminar or scientific conference and receiving mostly blank stares were long gone. Now, CRISPR seemed to be on everyone’s lips and the topic of every conversation. And yet it was still only the tip of the iceberg.

Instead of remaining an unwieldy, uninterpretable document, the genome would become as malleable as a piece of literary prose at the mercy of an editor’s red pen.

If we changed the twenty-letter RNA code to match the sequence of a specific human gene and then transplanted Cas9 and the new guide RNA into human cells, CRISPR would make a surgical cut in the targeted gene, marking that site for repair. By slicing the DNA apart, CRISPR would be acting like a red alert triggering the cell to fix the damage, but in a way that we could control.

Humans (all eukaryotic organisms, for that matter) constantly suffer DNA damage—it occurs when we are exposed to carcinogens, UV light, or x-rays, for example—and

that fix double-strand breaks. Thus, in the most basic scenario, if CRISPR succeeded in cutting a gene, the cell would respond by simply gluing the DNA back together, much like welding two pieces of metal pipe together.

Much to my delight, the first few weeks of 2013 were marked by the publication of a whopping five articles on CRISPR besides our own, all describing similar kinds of experiments in which the system had been used to edit genes in cells, just as we had proposed in 2012.

In addition to editing genes in embryonic kidney cells, CRISPR had been programmed to slice up DNA in human leukemia cells, human stem cells, mouse neuroblastoma cells, bacterial cells, and even one-cell embryos from zebrafish, a popular model organism for genetics studies. CRISPR wasn’t just showing some signs of success; it was exhibiting incredible versatility.

As long as the Cas9 protein was present and the guide RNA had a twenty-letter code that matched a twenty-letter DNA code, it seemed that virtually any gene in any cell could be targeted, cut, and edited.

Witnessing the protein and RNA molecules naturally deployed as antiviral defenses in bacteria being used to snip apart and precisely edit DNA sequences across the animal kingdom was breathtaking.

A bout of publications in the fall of 2013 reported the successful use of CRISPR for gene editing in staples such as rice, sorghum, and wheat, and a year later, the list of plants had expanded to include soybeans, tomatoes, oranges, and corn.

In addition to simply slicing apart DNA and inserting new sequences into the target genome, they can now also deactivate genes, rearrange sequences of genetic code, and even correct single-letter mistakes, as Kiran Musunuru had demonstrated during my visit to his lab.

CRISPR can be described as a pair of designer molecular scissors because of its core function: to home in on specific twenty-letter DNA sequences and cut apart both strands of the double helix. Yet the types of gene-editing outcomes that scientists can achieve with this technology are remarkably diverse. For this reason, it might be better to describe CRISPR not as scissors but as a Swiss army knife, a tool with a panoply of functionalities that all stem from the action of a single molecular machine.

Herein lies the most basic power of CRISPR—it can destroy a gene’s ability to produce a functional protein. If a CRISPR-edited gene ends up with a small insertion or deletion, the corresponding mRNA produced from that gene will be similarly perturbed. And the majority of the time, those extra or missing letters will disrupt the strict three-letter grouping of genetic code, so the protein will be wildly mutated or, more commonly, not produced at all. In any case, the protein can’t play its normal role. Geneticists refer to this as a gene knockout, or KO, just like in boxing, since the gene’s function has effectively been shut off.

Fortunately, cells possess the machinery to perform a second type of repair, one that is far more precise and controlled than merely gluing broken DNA back together. Instead of joining DNA segments unrelated in sequence, this alternative mode—a pathway that early gene-editing researchers used to their advantage—exclusively rejoins segments that are similar in sequence. This pickiness explains the two synonymous terms that refer to this process: homologous recombination and homology-directed repair.

Armed with the complete CRISPR toolkit, scientists can now exert nearly complete control over both the composition of the genome and its output. Whether that’s done through sloppy end joining or precise homologous recombination, by one cut or multiple cuts or even no cuts at all, the range of possibilities is immense.

It’s become something of an old saw in our young field: what used to require years of work in a sophisticated biology laboratory can now be performed in days by a high school student. Some experts have suggested that, with today’s tools, anyone can set up a CRISPR lab for just $2,000.

Scientists the world over have already begun using CRISPR on other species in ways that defy imagination, and it won’t be long before human genomes are given the same treatment.

TOMATOES THAT CAN SIT in the pantry slowly ripening for months without rotting. Plants that can better weather climate change. Mosquitoes that are unable to transmit malaria. Ultra-muscular dogs that make fearsome partners for police and soldiers. Cows that no longer grow horns.

These organisms might sound far-fetched, but in fact, they already exist, thanks to gene editing.

In a sign of the kinds of aesthetic changes to animals that are now possible, companies have used gene-editing technologies to create new designer pets, such as gene-edited micropigs that never grow larger than small dogs.

This human-influenced evolutionary process—natural mutation followed by artificial selection rather than natural selection—is how agriculture has developed for millennia. As pioneering agriculturist Luther Burbank remarked in a speech in 1901, species weren’t fixed and unchangeable but rather “as plastic in our hands as clay in the hands of the potter or colors on the artist’s canvas, and can readily be molded into more beautiful forms and colors than any painter or sculptor can ever hope to bring forth.”

Scientists have used CRISPR to edit the genome of sweet oranges, and a team of California researchers is now attempting to apply the technology to save the U.S. citrus industry from a bacterial plant disease called huanglongbing—a Chinese name that translates as “yellow dragon disease”—that has devastated parts of Asia and now threatens orchards in Florida, Texas, and California.

Recently, food scientists at a Minnesota company called Calyxt used the TALEN gene-editing technology to alter two soybean genes, generating seeds with a drastic reduction in the unhealthy fatty acids and an overall fat profile similar to that of olive oil. They accomplished this without causing any unintended mutations and without introducing any foreign DNA into the genome.

Or will gene-edited crops suffer the same fate as GMOs, another type of genetically altered food and one that has met with incredible and, I would argue, misinformed opposition despite its vast potential for good?

well over fifty GMO crops have been developed and approved for commercial cultivation in the United States, among them canola, corn, cotton, papaya, rice, soybean, squash, and many more. In 2015, 92 percent of all corn, 94 percent of all cotton, and 94 percent of all soybeans grown in the United States were genetically engineered in this way.

The altered crops offer considerable environmental and economic advantages. By planting crops that have enhanced abilities to protect themselves against pests, farmers can attain higher yields while reducing their reliance on harsh chemical pesticides and herbicides.

Despite these benefits, and despite the fact that hundreds of millions of people have consumed GMO foods without any issues, these foods remain the target of vociferous criticism, intense public scrutiny, and strident protest, most of it without merit.

GMOs have been subjected to some of the most careful regulatory review of any human consumables on the market, and there is near-unanimous consensus that GM food is every bit as safe as conventionally produced food. GMOs have received support from federal regulators in the United States, the American Medical Association, the U.S. National Academy of Sciences, the Royal Society of Medicine in the UK, the European Commission, and the World Health Organization. Nevertheless, nearly 60 percent of Americans perceive GMOs as unsafe.

gene-edited organisms are often no different than those organisms produced by mutation-inducing chemicals and radiation. Furthermore, scientists have used methods to avoid leaving any traces of CRISPR in the plant genome once the gene-editing task is complete.

At the moment, new genetically modified crops face a confusing array of regulatory hurdles, with jurisdiction split among the Food and Drug Administration, the Environmental Protection Agency, and the U.S. Department of Agriculture. The approval process is both long and expensive and includes what some consider to be an unfair and onerous set of requirements.

Biotechnology can help us shore up our food security, stave off malnutrition, adapt to climate change, and prevent environmental degradation around the world. This progress will remain out of reach, however, until scientists, companies, governments, and the public at large work together to make it happen. Each of us can contribute to this partnership in a very basic way. It starts with an open mind.

The first genetically engineered animal to be approved for human consumption in the United States—a fast-growing GMO salmon breed, called AquAdvantage—made it to market only after a twenty-year battle with FDA regulators and at a cost of over eighty million dollars for its developer. The gene-spliced salmon contains an extra growth hormone gene, resulting in a fish that reaches market weight in half the time of a conventionally farmed salmon and without any changes to its nutritional content or any increased health

risks for either the fish or the humans who eat it.

There, the pigs were exposed to some one hundred thousand viral particles and continually monitored. Remarkably, the gene-edited pigs remained completely healthy and free of any traces of virus. This strategy—saving pigs from viruses by knocking out the genes that viruses depend on—has been so effective that it’s already being adopted by other researchers to reduce suffering and waste in other corners of the meat industry.

Not all cows have horns, though. In fact, many beef cattle breeds—including the popular Angus—are naturally horn-free. In 2012, a German research team discovered the exact genetic cause: a complex mutation involving the deletion of 10 DNA letters and the insertion of 212 DNA letters on chromosome 1. Inspired by this knowledge, the scientists at Recombinetics used gene editing to copy the exact same change into the genome of blue-ribbon dairy bulls, creating cattle whose prized genetics—crafted over centuries of selective breeding for optimal milk production—weren’t otherwise altered. The first such animals to be born, two hornless dairy calves named Spotigy and Buri, will never know the horror of having their horn buds removed.

The hornless cattle might have been produced by years of conventional breeding. Gene editing merely allowed the same outcome to be achieved much more efficiently. If CRISPR and related technologies can eliminate inhumane practices like dehorning, reduce antibiotic usage, and protect livestock from deadly infections, can we afford not to use them?

Humans have been changing the genetic makeup of plants and animals since long before the advent of genetic engineering. Should we refrain from influencing our environment with this new tool even though we haven’t showed such restraint in the past? Compared to what we’ve done to our planet already, whether intentional or not, is CRISPR-based gene editing any less natural or any more harmful? There are no easy answers to these questions.