Hacking Darwin

by Jamie Metzl

  This technology was soon used to edit the genomes of mice, rats, cows, pigs, and other nonhuman mammals in all sorts of laboratory experiments with far greater precision than ever before. ZFN quickly became the predominant gene-editing technology in research labs around the world. Its predominance didn’t last long.

  In 2011, an even more convenient gene-editing tool was discovered. Transcription activator-like effector nucleases, or TALENs, was yet another obscurely named but revolutionary tool. TALENs also made double cuts to the DNA ladder but was more flexible and versatile than ZFN and could be used to edit a broader range of genetic target sites with greater specificity.

  In those ancient times of just a few years ago, TALENs seemed like magic. It was used to model multiple human diseases more efficiently and effectively, and to create genome-edited mice, cattle, pigs, goats, sheep, and even monkeys. As the process improved, it was used to eliminate a genetic eye disease in mice, and its prospects for helping cure human diseases seemed extremely promising. Recognizing its significance, the influential journal Nature Methods named TALENs its “Method of the Year 2011.”1 But while TALENs was easier to use than ZFN and seemed relatively superfast at the time, it too was nothing compared to the world-changing gene-editing tool in the making for a quarter of a century or billions of years, depending on how you are counting, but racing around the corner.

  This new tool begins with among the smallest of organisms.

  Bacteria are one of the earliest life forms on earth and the ultimate survivors. Viruses have been attacking bacteria for more than a billion years in their never-ending quest to find hosts into which to insert a tiny package of viral DNA. The viruses aren’t doing this out of malice. Hijacking and transforming host cells into tiny virus-producing machines is the virus’s sole strategy for survival. The viruses are aggressive, but the bacteria wouldn’t have thrived for so long if they didn’t build their own defensive survival strategy along the way.

  In 1987, researchers from Japan’s Osaka University examining a sequence of chromosomal DNA observed a series of genetic code repeats in a series of code clusters. A couple of year later, a young Spanish researcher named Francisco Mojica, who was studying sequenced bacteria with extreme salt tolerance, kept seeing the same types of repeated clusters of palindromic (e.g., madam I’m Adam) code popping up in the bacterial DNA.

  When Mojica compared the sequences he’d uncovered with those other researchers had placed in the common GenBank database, he began to notice that the bacteria’s palindromic repeated code clusters matched some of the same code clusters in certain viruses. At that time, no one knew what these clusters of code were for or if they were even important. Mojica and other researchers like Giles Vergnaud and Alexander Bolotin in France, however, made a series of brilliant educated guesses that the bacteria were using the code repeats as some type of immune system.2 A Dutch researcher, Ruud Jansen, later named these sequences clustered regularly interspaced short palindromic repeats. The name was a mouthful, so he shortened it into the more user-friendly acronym CRISPR.

  Around the same time, scientists at Danisco, the largest yogurt company in the world, learned about Bolotin’s work. Because the Streptococcus thermophilus bacteria is a mainstay in the process of turning milk into yogurt, Danisco scientists Philippe Horvath and Rodolphe Barangou started wondering if understanding how that bacteria responded to a viral attack might suggest new ways of preventing their yogurt and cheese cultures from occasional collapse.

  Using the lessons learned from Mojica, Bolotin, and others, Horvarth and Barangou exposed their culture bacteria to viruses, killing most of the bacteria. But when they repeatedly cultured the surviving bacteria and introduced the same viruses, the bacteria got progressively better at fighting them off. The bacteria had, in effect, developed an immunity to the viruses just like we get immunity to chickenpox after an exposure—just as Vergnaud and Bolotin had predicted. Understanding where these CRISPR repeats came from and how they functioned forced researchers to look far back into the deep history of microbial life on earth.

  Although the battle between virus and bacteria had been going on for a long time, scientists didn’t know much about how it was playing out until new genome-sequencing tools made a deeper level of scrutiny possible. The discovery of CRISPR came at the intersection of genome sequencing and big-data analytics. The “heroes of CRISPR” were code-breakers cracking the genetic code for how the bacteria defended itself.

  A CRISPR is like an Old West most-wanted poster of a virus that a bacterium stores in its own genetic code after an initial exposure. The bacteria archives fragments of the viral DNA from these past exposures into the bacteria’s own genetic code to create a series of genetic “mug shots” of bad guy viruses.

  If the virus rolls into cellular town, the bacteria sends an RNA probe to search for code in the virus’s DNA that matches the stored genetic CRISPR target list. When it finds one, the bacteria uses an enzyme to bind to the virus’s matching code and cut apart the viral DNA just at the site where the bacterial and viral codes match. When this works, the viral attackers are cut to pieces, the bacteria survive the attack, the piano starts up again, and the saloon customers go back to their card games.

  But this story isn’t just about viruses and bacteria. Because some bacteria merged into the cells of most other life forms hundreds of millions of years ago, the genetic code that originally stemmed from the ongoing war between viruses and bacteria became replicated in nearly all cells across the spectrum of life. CRISPR, it turned out, held the key to potentially editing the code of all life and changing biological life as we know it.

  As more researchers began to explore the science of CRISPR, big strides in understanding this remarkable new tool came fast and furious. In 2010, Sylvain Moineau and his colleagues showed how the CRISPR-Cas9 (for CRISPR-associated gene number nine) system made double-stranded breaks in DNA at predictable and precise locations. The following year, Emmanuelle Charpentier figured out how small bits of two different types of RNA guide the Cas9 enzyme to its target.

  Then the following year, in 2012, Charpentier and her new partner, Berkeley biochemist Jennifer Doudna, as well as Martin Jinek, cleverly adapted the CRISPR-Cas9 system into a precise tool that could be harnessed to cut any strand of DNA. They also figured out how the system could be used to insert additional new DNA. After being cut, the DNA tries to rebind where the cut has been made and will grab the available DNA researchers have positioned to fit into the opening. This made the gene-editing process far easier than ever before. The following year, Doudna, Charpentier, and Harvard/MIT researcher Feng Zhang announced that CRISPR-Cas9 could be used to target multiple locations in the human genome at once.

  If we had to summarize CRISPR into a single sentence, it would be this: CRISPR systems deploy the same tiny scissors bacteria use to cut up attacking viruses to snip any genetic code in a targeted place and potentially insert new genetic code. The following chart provides another helpful overview.

  EDITING A GENE USING

  THE CRISPR/CAS9 TECHNIQUE

  1Scientists create a genetic sequence, called a “guide RNA,” that matches the piece of DNA they want to modify.

  2This sequence is added to a cell along with a protein called Cas9, which acts like a pair of scissors that cut DNA.

  3The guide RNA homes in on the target DNA sequence, and Cas9 cuts it out. Once their job is complete, the guide RNA and Cas9 leave the scene.

  4Now, another piece of DNA is swapped into the place of the old DNA, and enzymes repair the cuts. Voilà, you’ve edited the DNA.

  Source: Business Insider.

  The CRISPR-Cas9 system is such a big deal because it has huge advantages over the older gene-editing approaches. While the ZFN and TALENs are bespoke systems, taking months to design, the CRISPR technology is almost entirely always the same, takes less than a few days to set up, and costs relatively little. But despite these overwhelming strengths, the CRISPR system has its shortcomings. Slici
ng the double strand of DNA using CRISPR was significantly more precise than with ZNF or TALEN, but such an aggressive cut opened the door for even greater unintended effects.3*

  While the scientific world and the popular media were swooning over CRISPR-Cas9, a steady stream of advances was making abundantly clear both that CRISPR-Cas9 was even more versatile than previously understood and that it was not the last word in precision gene editing but just the beginning.

  Rather than an equivalent to scissors, the CRISPR system is now looking more like a versatile Swiss Army knife able to record genetic changes within a cell over time, identify specific virus strains, test for infections, spatially reorganize the genome, and perform a host of other functions.4 Another approach pioneered by the Broad Institute’s Feng Zhang pairs CRISPR with a different enzyme, Cas13, to edit the messenger RNA to help better target where cuts and changes to the genome can be made.

  Scientists are also now using CRISPR not just to change the genes but also to alter the epigenetic marks dictating how the genes are expressed.5 Although early skeptics of human gene editing correctly warned that epigenetic influences on the expression of genes made effective gene editing a lot more complicated than first understood, recent advances have made clear that epigenetic editing is “on the verge of reprogramming gene expression at will.”6 As the rate of change in gene editing is accelerating, the process is being made cheaper and more precise, and the globally networked community of scientists is sharing ideas at a level and speed that would have been unthinkable not just to a relatively isolated monk like Gregor Mendel but also to some of the world’s most sophisticated scientists only a decade ago.

  The step-by-step progression from basic plant and animal experiments and applications to increasingly substantial human uses is already underway.

  The first phase of normalization of precision gene editing involves using these tools to advance basic research. One of the most significant early benefits of CRISPR-Cas9 is its ability to target and isolate for study specific DNA sequences. By using the “molecular scissors” of Cas9 enzyme to quickly, easily, and cheaply cut a specific DNA sequence, scientists can study the effect of a compromised gene on cells and organisms. This is a huge deal for scientific research that is increasingly being translated into real-world applications on plants, laboratory and farm animals, and preliminarily humans.

  A plant pathologist at Pennsylvania State University, for example, used CRISPR to target a whole family of genes that encodes polyphenol oxidase (PPO), an enzyme responding to oxidization.7 The gene-edited “Arctic apple” can be sliced and left out in the open without browning because scientists have used CRISPR to silence a gene controlling production of an enzyme that causes apples to brown.8 A virus-resistant Rainbow papaya gene edited to avoid the devastating papaya ringspot virus is already in supermarkets, as is the bruise-resistant Innate potato. Del Monte has received approval to gene-edit a pink pineapple modified to contain more of the antioxidant lycopene than regular pineapples. A waxy corn that makes better cornstarch, wheat with a higher fiber and lower gluten content, tomatoes better able to grow in warm climates, and camelina with enhanced omega-3 fatty acids are all on the way, thanks to CRISPR technology. In our yogurt and in plants like these, we humans are already bringing CRISPR-edited genes into our bodies.

  Gene editing crops isn’t just about keeping our mushrooms from browning and our potatoes from bruising. This technology also has the potential to protect billions of dollars of crops and save the lives of millions of the world’s poorest people.9 In Africa and South Asia, where huge percentages of the population are subsistence farmers, average temperatures are warming, and populations are expanding, this need is particularly stark. Using gene editing to create new, more resilient, faster-growing varieties of rice and other crops that need less water could, in the words of Microsoft founder and philanthropist Bill Gates, “be a lifesaver on a massive scale.”10

  Gene-editing tools are also being used aggressively on animals. In addition to generating the gene-edited laboratory rats and mice for biological research, CRISPR is being used to alter the genes and gene expression of a menagerie of other animals. The Gates Foundation, for example, is supporting efforts by the Global Alliance for Livestock Veterinary Medicines to create genetically modified “supercows” that can withstand very hot temperatures while producing much more milk than traditional cows.11 One scientist whose daughter is allergic to eggs is using CRISPR-Cas9 to engineer hypoallergenic chicken eggs. Researchers are using CRISPR to genetically engineer virus-resistant and faster growing pigs and parasite-resistant and hornless cattle, which could save commercial ranchers hundreds of millions of dollars per year.

  On a less practical level, China’s BGI-Shenzhen created micropigs designed to be lab animals or pets that grow to a maximum of 33 pounds, one seventeenth of the weight of an average adult pig.12 Harvard’s George Church is even exploring the possibility of using CRISPR and industrial-scale multiplex automated genome engineering tools to revive the extinct woolly mammoth by simultaneously altering multiple genes of Asian elephant embryos.13

  Wooly mammoths notwithstanding, accommodating gene-edited plants and animals in our food supply and homes will create a greater level of acceptance of gene editing more generally. The incredible promise of gene-editing tools to treat and cure diseases will propel social acceptance of the technologies even more.

  Although this book is about the heritable genetic alterations that will ultimately transform our species, the path from here to that future will pass through the application of nonheritable genetic therapies to treat diseases and improve health care. Transforming our evolutionary process is the ultimate destination of the genetics revolution, but medicine will be an essential way station on the road from where we now are to where we are inevitably going.

  When the possibility of altering a living human’s genes to treat disease was introduced in the 1980s, it was recognized as a potential but far-off game-changer. Over the ensuing years, the possibility of manipulating genes to prevent or treat multiple diseases has become an increasingly real possibility. Instead of the more traditional methods of treating diseases with surgery or drugs, gene therapies seek to use genes to treat or prevent disease by knocking out or tuning off a mutated gene, replacing a mutated gene with a healthy copy of the same gene, and/or adding a new gene to help the body fight a particular disease.* As exciting as all this sounds and is, the path toward realizing gene therapies in health care has been a rocky one.

  By 1999, doctors at the University of Pennsylvania were confident the science of gene therapy had advanced enough that it could be used to treat Jesse Gelsinger, an eighteen-year-old with ornithine transcarbamylase (OTC) deficiency, a rare genetic disorder that causes increased ammonia levels in the blood often leading to brain damage and premature death. Four days after receiving an infusion of a corrective OTC gene delivered in a manipulated cold virus, Gelsinger died.

  Jesse Gelsinger’s death was not just a personal and family tragedy, it also put a screeching halt to the application of gene therapies to treat disease in the America, then by far the leading venue for these technologies in the world. The FDA prohibited the University of Pennsylvania from continuing its human gene therapy trials, began investigating sixty-nine other ongoing gene therapy trials in the United States, and started requiring a far higher level of patient safety for gene-therapy trials.14

  But with the prospect of gene therapy slowed, researchers got to work developing better and safer gene transfer protocols.15 As gene-editing techniques improved in the ensuing years, so did the prospects for gene therapy. By 2009, the journal Science declared the “return of gene therapy” as the major breakthrough of the year,16 and the more recent development of better gene-editing tools like CRISPR have only made the future of gene therapies seem even brighter.17

  Although many gene-therapy protocols are now being actively explored, one of the most exciting and widely covered in the media is genetically enhancing the
ability of a person’s T cells, white blood cells that play an essential role in the body’s natural immune response. In CAR-T therapy, blood cells are extracted from the body of a person with specific cancers and then engineered to boost the ability of their T cells to express a chimeric antigen receptor (CAR) before being put back into the person’s body with cancer-fighting superpowers.

  In the first three months of clinical trials of this approach by the pharmaceutical company Novartis in 2017, 83 percent of patients showed a significant remission rate in their cancers. Billions of investment dollars are now flowing into scores of companies like Novartis, Gilead, Juno Therapeutics, Celgene, and Servier that are working on gene therapies for cancer, and many hundreds of clinical trials are running around the world. CAR-T therapy still has some significant challenges, and a small number of people have even died during clinical trials, but there is no doubt removing, editing, and reintroducing genes will play a greater role in fighting cancer and other diseases going forward. As of August 2018, the U.S. Food and Drug Administration had received more than seven hundred applications for gene therapy trials.18

  Removing cells from the body to edit and return them in gene therapy is a massive step forward, but an even bigger advance is our growing ability to use CRISPR and other tools to edit the offending cells still inside the body. Editing cells inside the body would open a whole new set of possibilities not only for treating disease but also potentially for gene editing human embryos.

  A recent study, for example, gene edited cells to treat diseased human livers grown inside living mice.19 Another study used CRISPR base editors to precisely correct genetic mutations causing a metabolic liver disease in adult mice.20 These types of in vivo gene therapies are not yet ready for prime time but is showing promising early results in potentially treating congenital blindness, hemophilia B, beta-thalassemia, Duchenne muscular dystrophy, cystic fibrosis, and spinal muscular atrophy. Incredible new companies like Editas, cofounded by George Church, Feng Zhang, and others, and Caribou Biosciences, cofounded by Jennifer Doudna, and others, are moving forward aggressively to develop new ways of treating multiple diseases with CRISPR gene editing. A forty-four-year-old man in California in November 2017 became the first person to have genes edited inside his body, in this case to treat the metabolic disease Hunter syndrome.