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

by Jennifer A. Doudna

“Someday we may consider it unethical not to use germline editing to alleviate human suffering.” This remark turned our conversation on its head, and it still comes to mind whenever I meet with parents or would-be parents who are facing the devastating effects of genetic disorders.

They concluded that a single cell in her body must have experienced an uncommon and usually catastrophic event called chromothripsis—a recently discovered phenomenon in which a chromosome suddenly shatters and is then repaired, leading to a massive rearrangement of the genes within it.

The human genome comprises about 3.2 billion letters of DNA, with around 21,000 protein-coding genes.

The human genome also includes a separate mini-chromosome—just sixteen thousand letters of DNA—located in mitochondria, the energy-producing batteries of the cell. Unlike the genetic code found in other chromosomes, mitochondrial DNA is inherited exclusively from the mother.

full 8 percent of the human genome—over 250 million letters of DNA—is a remnant of ancient retroviruses that infected ancestors of our species millennia ago.

These next-generation gene-editing systems had three critical requirements: They had to recognize a specific, desired DNA sequence; they had to be able to cut that DNA sequence; and they had to be easily reprogrammable to target and cut different DNA sequences.

CRISPR—referred to a region of bacterial DNA and that the acronym stood for “clustered regularly interspaced short palindromic repeats.”

every cell had a different CRISPR array due to the unique sequences interspaced between the repeats.

In 1923, d’Herelle helped Soviet scientists set up an institute in Tbilisi, present-day Georgia, dedicated to bacteriophage research; at its peak, the institute had over a thousand employees producing tons of phages a year for clinical use. Phage therapy has continued up to modern times in certain parts of the world—about 20 percent of bacterial infections are treated with phages in Georgia today—but

Bacteriophages aren’t just popular laboratory pets, though; they are also the most prevalent biological entity on our planet—by a long shot. They are as ubiquitous in the natural world as light and soil, and they can be found in dirt, water, our intestines, hot springs, ice cores, and just about anywhere else that supports life. Scientists estimate that there are somewhere on the order of 1031 bacteriophages on earth; that’s ten million trillion trillion, or a one with thirty-one zeros after it. A single teaspoon of seawater contains five times more phages than there are people in New York City.

Could a strain of S. thermophilus actually make itself more resistant to a particular bacteriophage by splicing new DNA into its own CRISPR region that matched the sequences of DNA found in the phage?

Sure enough, after isolating the genomic DNA from each mutant strain, the researchers found that every single CRISPR region had expanded to include a new snippet of DNA spliced between the repeats. Furthermore, these new spacers perfectly matched the DNA of the phage to which that strain was now immune. What made this apparent mode of immunity so remarkable was that, because these changes were physically embedded in the bacteria’s CRISPR DNA, the new immunity was heritable and would be passed down every time the bacterial cells reproduced.

Since I was still focused on Type I systems, I might not have been drawn into tackling this new line of research had it not been for a fortuitous encounter with a colleague whose lab was located halfway across the globe and whose work I had only read about.

Serendipity!

On June 8, 2012, a sunny Friday afternoon, I clicked Confirm on my computer, formally submitting our paper for consideration to the journal Science. It would be published just twenty days later, on June 28,

!!

Jaenisch’s team employed CRISPR to achieve the same feat in just one month, using a simple, streamlined protocol: microinjection of CRISPR components directly into one-cell embryos, followed by implantation of the gene-edited embryos into a female’s womb. Moreover, they showed that CRISPR could be programmed with not just one RNA guide, but multiple different guides, directing Cas9 to cut up and edit several DNA sequences in mouse embryos simultaneously. This kind of one-step, multiplex gene editing had never before been performed in mice.

This sloppy, error-prone process leaves telltale clues—short insertions or deletions of DNA (known as indels) flanking the sequence cut apart by CRISPR.

TYR mutations in humans lead to a deficiency in tyrosinase and cause type I albinism, a genetic condition associated with vision defects, pale skin lacking pigmentation, and red eyes.

One could also track how that efficiency changed over time as various laboratories optimized both the design and preparation of CRISPR. In the Texas study, only 11 percent of mice progeny were fully albino, and pictures of the litters revealed a salt-and-pepper pile of infant pups, with quite a bit more pepper than salt. Barely a year later, a Japanese research team repeated the same experiment but with a few minor tweaks and achieved efficiencies of 97 percent, with thirty-nine out of forty pups displaying a radiant, homogeneous albino exterior.

The possibilities of this technology for agriculture were highlighted in my mind when, in 2014, scientists at the Chinese Academy of Sciences used gene-editing tools, including CRISPR, to alter the six copies of the Mlo gene in Triticum aestivum, or bread wheat, one of the world’s most important staple crops. Plants that had all six mutated Mlo genes were resistant to powdery mildew, a fantastic result, and furthermore, the researchers didn’t have to worry about harmful or undesired effects of any other mutations because only the Mlo genes had been edited.

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. In South Korea, scientist Jin-Soo Kim and his colleagues hope gene editing in bananas can help save the prized Cavendish variety from extinction, an outcome threatened by the spread of a devastating soil fungus. And elsewhere, researchers are even toying with the possibility of inserting the entire bacterial CRISPR system, reprogrammed to slice up plant viruses, into crops, providing them with a completely new antiviral immune system.

Using gene editing, researchers at Calyxt easily addressed the problem in Ranger Russet potatoes: they inactivated the single gene that produced glucose and fructose. The result: a 70 percent drop in acrylamide levels in potato chips made with the enhanced spuds, and no chip browning.

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.

Almost everything we eat has been altered by humans, often by generating random mutations in the DNA of seeds used to breed plants with desired traits. Thus, the distinction between “natural” and “unnatural” has been obscured. Red grapefruits created by neutron radiation, seedless watermelons produced with the chemical compound colchicine, apple orchards in which every tree is a perfect genetic clone of its neighbors—none of these aspects of modern agriculture is natural. Yet most of us eat these foods without complaint.

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.

In the early 2000s, a Japanese team bred pigs containing a spinach gene that altered the way the animals metabolized fatty acids; the transgenic swine had a healthier fat profile, but the scientists’ work was widely condemned, and the pigs never made it out of the lab. Around the same time, a Canadian team created the Enviropig, an environmentally friendly transgenic pig containing an E. coli gene that allowed the animals to better digest a phosphorus-containing compound called phytate. Normal pig manure retains high phosphorus levels that leach into streams and rivers, causing algal blooms, the death of aquatic animals, and the production of greenhouse gases; Enviropig manure contained 75 percent less phosphorus,

Cattle farmers have known about double muscling for years because of its high frequency in two popular cattle breeds: the Belgian Blue and the Piedmontese. These cows have 20 percent more muscle on average, a higher meat-to-bone ratio, less fat, and a higher percentage of desirable cuts of meat, making them a beef producer’s dream. In 1997, three labs determined that a single gene was responsible for this exceptional form of muscle development. The gene, called myostatin, behaves like a natural brake on the body’s production of muscle tissue. The two breeds of cattle these labs were studying have different kinds of mutations—Belgian Blue cows are missing eleven letters of DNA, whereas Piedmontese cows have just a single-letter mutation—but in both cases, the protein product of the myostatin gene is defective.

There has never been a greater need for new transplantation options. In the United States alone, more than 124,000 patients are currently on the waiting list for transplants, yet only approximately 28,000 procedures are carried out annually. It’s been estimated that a new individual is added to the national transplant list every ten minutes and that an average of twenty-two people a day die while waiting for a transplant

Unlike micropigs, whose health is no different than their normal-size relatives, extensive inbreeding of dogs has had devastating health consequences. Labradors are prone to some thirty genetic conditions, 60 percent of golden retrievers succumb to cancer, beagles are commonly afflicted with epilepsy, and Cavalier King Charles spaniels suffer from seizures and persistent pain due to their deformed skulls. These poignant medical problems haven’t kept humans from letting tastes dictate the genotype and phenotype of humankind’s best friend.

In the summer of 2014, George Church’s team at Harvard, led by Kevin Esvelt, proposed a way to design and build gene drives with the help of efficient gene editing. In essence, the idea relies on a gene knock-in approach, in which scientists use CRISPR to cut DNA at an exact location and insert a new sequence of letters into the breach. There is one major difference with a gene drive, however: part of the new DNA added in contains the genetic information that encodes CRISPR itself. Like that sci-fi trope of a self-replicating machine, a CRISPR gene drive can autonomously copy itself into new chromosomes, allowing it to grow exponentially within a population. By combining CRISPR with various genetic payloads, such as pathogen-resistance genes, Esvelt theorized, scientists could program CRISPR to copy not only itself, but any other desirable DNA sequences.

It’s not the first time that scientists have turned to genetic engineering to reduce insect populations. A common practice used for decades involves the release of sterilized males into the environment; the technique has all but eliminated certain agricultural pests through North and Central America. Another approach being developed by a British company called Oxitec involves inserting a lethal gene into the mosquito genome, and field trials have already commenced in Malaysia, Brazil, and Panama. However, these strategies are inherently self-limiting; the genetic alterations are rapidly eliminated by natural selection, and the only way to make a dent in mosquito populations is to repeatedly release large batches of the modified insects.

But the most momentum, by far, is in the push to use gene drives to target the mosquito. The mosquito causes more human suffering than any other creature on earth. Mosquito-borne diseases—malaria, dengue virus, West Nile virus, yellow fever virus, Chikungunya virus, Zika virus, and many others—have an annual death toll in excess of one million.

In December of 2013, less than a year after several labs, including my own, reported the successful use of bacteria-derived CRISPR molecules in human cells for gene editing, a team of Chinese researchers programmed the same CRISPR molecules to find and fix a single-letter mutation among the 2.8 billion DNA letters of the mouse genome. In so doing, they performed the first outright, CRISPR-based cure of a genetic disease in a live animal.

Some scientists are employing CRISPR in human cells to block viral infections, just like this molecular defense system naturally evolved to do in bacteria. In fact, the first clinical trials to use gene editing are aimed at curing HIV/AIDS by editing a patient’s own immune cells so the virus can’t penetrate them. And in another landmark effort, the first human life was saved by gene editing in combination with another emerging breakthrough in medicine: cancer immunotherapy, in which the body’s own immune system is trained to hunt down and kill cancerous cells.

Two of the promising disease targets for ex vivo CRISPR therapies are sickle cell disease and beta-thalassemia.

If doctors can isolate stem cells from a patient’s bone marrow, repair the cells’ mutated beta-globin genes with CRISPR, and then return those edited cells to the patient, they won’t have to worry about donor availability or the risk of an immunological clash between the patient’s body and the transplanted cells.

Believe it or not, some lucky people are naturally resistant to HIV. These individuals lack thirty-two letters of DNA in the gene for a protein called CCR5, which is located on the surface of white blood cells—those cells that form the bedrock of the body’s immune system. CCR5 proteins are one of the parts of the cell’s surface that the HIV virus latches onto in the initial stage of its invasion. This specific, thirty-two-letter deletion causes the CCR5 protein to be truncated and prevents it from making its way to the cell surface. Without CCR5 proteins to attach to, HIV molecules can’t infect the cells. In people of African and Asian descent, the thirty-two-letter CCR5 deletion is virtually nonexistent, but it’s fairly prevalent among Caucasian people; 10 to 20 percent of Caucasians possess one copy of the mutated gene, and homozygous individuals—those who possess two copies—are completely resistant to HIV.

But recent studies have demonstrated conclusively that we can accomplish the same thing—that is, prevent HIV from latching onto CCR5—by editing the CCR5 gene itself. Multiple labs have already pulled this off using CRISPR, at least with cells in a petri dish. But credit for the first success in editing the CCR5 gene in human subjects goes to the ZFN technology and to a California-based company called Sangamo Therapeutics.

One vector—the generic scientific term for a carrier of genetic information—has been an especially big asset to researchers developing in vivo gene-editing therapies: an innocuous human virus known as adeno-associated virus (AAV).

Another encouraging aspect of AAV is its sheer natural diversity. By isolating different strains of the virus and then mixing and matching them in different ways, researchers have assembled a family of AAV vectors that can target cells in many different types of tissues. One strain of AAV might be best suited to deliver CRISPR to cells of the liver, while another might work best in the central nervous system, the lungs, the eyes, or the cardiac and skeletal muscles.

By the end of 2015, no fewer than four independent laboratories delivered CRISPR to fully grown mice suffering from muscular dystrophy and showed that the ravages of the disease could be reversed. By packaging genetic instructions for CRISPR into AAV vectors, the researchers repaired skeletal and cardiac muscle cells, either by injecting the loaded viruses into the mouse’s muscles or by delivering the viruses to the same tissues through the bloodstream.

Rather than being a form of treatment in and of itself, CRISPR is advancing cancer care as a tool and a support system for existing therapies. It is expanding our understanding of cancer biology, and it is also accelerating immunotherapy treatments, which harness the body’s own immune system to fight cancer.

For example, in one study from Harvard Medical School, Benjamin Ebert’s team of researchers wanted to understand the genetic causes of acute myeloid leukemia, a cancer of white blood cells. By programming CRISPR to edit different genes—using a different guide RNA to match each one—they set out to knock out eight candidate genes. This kind of multiplexed gene editing would have been unthinkable before, but with CRISPR, it was straightforward. After editing the genes in blood stem cells in all different combinations and then injecting the cells back into the bloodstream of live mice, the researchers waited to see which animals came down with acute leukemia (a sort of reverse ex vivo application). Then, by cross-checking those animals with the genes that had been inactivated with CRISPR, Ebert’s team deduced the exact constellation of gene mutations necessary and sufficient to promote leukemia. Experiments like these are proving invaluable for advancing human cancer research.

In the weeks that followed the cell transfer, a miraculous transformation took place in the one-year-old girl; her leukemia began responding to the edited T cells. When her health improved enough, Layla underwent another bone marrow transplant, and within months, the cancer was in complete remission. What had begun as a major gamble—attempting a treatment that had up until that point been tested only in mice—ended up a resounding success and a major endorsement for using gene editing to further immunotherapy.

There’s a third strategy for avoiding potential off-target mutations, one with which scientists have already made great headway: engineering CRISPR to be more discriminating in how it recognizes the target DNA. For example, scientists have successfully expanded the sequence of DNA that CRISPR has to recognize, minimizing the chances of an unlucky mismatch—a strategy not unlike increasing the length of a computer password to reduce the likelihood that someone can guess

preimplantation genetic diagnosis, or PGD.

The implications of this kind of genetic testing are extreme, yet it’s not even the latest or most advanced technology associated with assisted reproduction. That distinction goes to mitochondrial replacement therapy, colloquially known as three-parent IVF.

Every person experiences roughly one million mutations throughout the body per second, and in a rapidly proliferating organ like the intestinal epithelium, nearly every single letter of the genome will have been mutated at least once in at least one cell by the time an individual turns sixty.

Even the sex cells that create the embryo—the mother’s egg and the father’s sperm—have incorporated new mutations that never before existed in either family’s germline. As a result, each one of us begins life with fifty to a hundred random mutations that arose de novo (“anew”) in our parents’ germ cells.

But plenty of other genetic enhancements do result from simple mutations that could be re-created with CRISPR. For example, mutations in the EPOR gene, which responds to the hormone erythropoietin (the famous doping drug used by Lance Armstrong and countless other athletes), confer exceptional levels of endurance; mutations in a gene called LRP5 endow individuals with extra-strong bones; mutations in the MSTN gene (the same myostatin gene that’s been edited to create supermuscular pigs and dogs) are known to result in leaner muscles and greater muscle mass; mutations in a gene called ABCC11 are associated with lower levels of armpit odor production (and, oddly, the type of earwax an individual produces); and mutations in a gene called DEC2 are associated with a lower requirement of daily sleep.

Plenty of other potential applications for germline editing blur the line between therapy and enhancement. Editing CCR5 with CRISPR could confer lifelong resistance to HIV; editing the APOE gene could lower an individual’s risk of developing Alzheimer’s disease; altered DNA sequences in IFIH1 and SLC30A8 could lower a person’s risk of developing type 1 and type 2 diabetes; and changes to the GHR gene could reduce an individual’s risk of cancer.