It’s hard to think of a technology that has as swiftly and profoundly changed a vast range of scientific fields as CRISPR-Cas9. It is so cheap, radical, and revolutionary that its use has pumped entire fields of investigation into instant overdrive. Just over three years ago, scientists at UC Berkeley and MIT separately discovered a system that bacteria have used for billions of years to rid their DNA genomes of invading mini-viruses, called phages. The scientists fine-tuned it in the lab, finding ways to use the bacterial gene-chopping system on animal and human cells. They touched off a frenzy that’s affecting everything from biotech stock prices to genetic modification of disease-carrying mosquitoes; drug development to the resurrection of long-extinct species of plants and animals. The genetic technology that is upending every field of biology, medicine, and pharmaceutics poses not only exciting opportunities for discovery, but also profound moral, ethical, and foreign policy questions.
CRISPR stands for “clustered regularly interspaced short palindromic repeats,” and Cas9 refers to “CRISPR-associated protein number nine.” The former was accidentally discovered by Japanese researchers in 1987 in their scrutiny of the bacterium E. coli, a sometimes pathogenic microorganism that is ubiquitous in humans and higher animals. At various positions throughout the bacterium’s genome, the Japanese found, were clusters of repeated DNA sequences that didn’t seem to mean anything. Even more mysteriously, further along the DNA at the end of a gene, the Japanese found there were always palindromic opposites to the first odd cluster. The two repeated clusters, or CRISPRs, looked like bookends, and appeared in sets surrounding genes all over the bacterium. Over the subsequent twenty-five years, scientists found these mysterious CRISPR palindromes in the DNA of every creature they studied, including throughout the human genome.
"It is an elegant, amazingly accurate gene editing method that bacteria have used for billions of years, but humans have only understood and deployed for about 36 months."
The landmark discovery made in 2012 by Jennifer Doudna at UC Berkeley in collaboration with Emmanuelle Charpentier, now with the Max Planck Institute for Infection Biology in Berlin, was that bacteria also contain special bits of RNA that mirror those CRISPR DNA bookends and attach like Velcro to specific CRISPR targets. The Velcro-like RNA bits are guides that direct scissor-like enzymes to designated spots to chop out genes. Those bizarre bookend CRISPRs turn out to be signposts, indicating where a particular gene starts and stops in the long DNA chain. And the Velcro-like RNA bits each mirror a particular CRISPR segment, drag a Cas9 enzyme into position, and snip: The DNA chain is cut at precisely the correct points to excise a gene. It is an elegant, amazingly accurate gene editing method that bacteria have used for billions of years, but humans have only understood and deployed for about thirty-six months. Bacteria use CRISPR to eliminate excess genes, and (in a sort of crude immune system) identify and destroy DNA inserted by invading phages. In 2013, Feng Zhang’s team at MIT’s Broad Institute announced the creation of a refined technique for using CRISPR-Cas9 to edit and chop mouse and human genes. (Berkeley and MIT are now battling patent rights in court.)
A Cure for HIV/AIDS?
Now that humans have figured out what microorganisms have been doing for a couple of billion years, scientists are going CRISPR-crazy, chopping DNA in and out of genomes ranging from tiny viruses to monkeys and people. On March 22, researchers from Temple University announced the successful use of CRISPR-Cas9 in test tubes to chop HIV viral genes out of latent hiding places inside human DNA—a first step on the road to possibly curing AIDS. (Another lab two weeks later showed that HIV can overcome the CRISPR-chopping, but the bad news only served to stimulate more experiments in a race to finally cure AIDS.) This comes on the heels of a Duke University team’s use of CRISPR-Cas9 in experimental mice to correct the mutation responsible for the rare human genetic disease Duchenne’s muscular dystrophy. The Duke group hitched the RNA and Cas9 onto a virus, injected it into live mice, and watched their muscles normalize thanks to the removal of “bad” genetic material, which allowed the good genes to be expressed and to make proteins muscles require. Next step: humans.
But CRISPR-Cas9 technology is about a lot more than curing diseases. A host of modifications of the technology nature created have allowed scientists to design genetically artificial microorganisms, mice, and rats that bear a range of traits, directly manipulate living cells, and rapidly find genes in human chromosomes. The CRISPR experiments are pouring forth from thousands of laboratories worldwide, with gene editing already executed that may render chicken eggs safe for people who are usually allergic to poultry eggs; create a possible way to save honey bees; turn pigs into safe organ donors for people; make animals into “farmaceutical” factories, generating products useful medically in other animals, even people; potentially bring back from extinction mastodons, passenger pigeons, wooly mammoths, even the dodo bird. Prevent inherited form of blindness in people? Almost there, according to a recent study.
In February, the UK government approved the use, under certain ethical limitations, of CRISPR-Cas9 to alter human beings, allowing researchers to manipulate human embryos to improve IVF fertilization success rates. While warning against altering heritable human genes, a UN bioethics committee applauded the UK decision as "a watershed in the history of medicine," noting that CRISPR-Cas9 “is unquestionably one of the most promising undertakings of science for the sake of all humankind." The merely advisory UN committee did call, however, for a ban on all CRISPR work aimed at altering the inherited human gene line, or creation of so-called “designer babies.”
The ethics, morality, and politics of CRISPR have quickly entered territory familiar to those that have engaged in prior debates over the deliberate genetic alteration of flu viruses, use of do-it-yourself biology labs to generate novel microorganisms, and sovereign “ownership” of pathogenic viruses. No country has yet adopted CRISPR-specific regulations, and no two nations seem able to agree about what is right, wrong, possible, or absolutely taboo in the brave new world of biology. Two years ago, many Western observers gasped when a Chinese team announced the use of CRISPR to genetically alter monkeys, passing multiple manmade traits onto their mutant offspring.
"No country has yet adopted CRISPR-specific regulations, and no two nations seem able to agree about what is right, wrong, possible, or absolutely taboo."
Later, researchers at Sun Yat-Sen University in Guangzhou announced successful CRISPR-Cas9 editing of human germline cells (sperm, ovaries, eggs, and the very first phase of dividing pre-fetus undifferentiated formations). Using what they termed “nonviable” human embryos from a fertility clinic, the Chinese team tried to reverse the genetic blood disease beta-thalassemia. Most of the eighty-six embryos didn’t survive the experiment, and only a quarter successfully underwent the CRISPR-Cas9 alteration, reversing the blood disease. Among those “successes” were apparently randomly made CRISPR cuts that produced undesired mutations. At best, the experiment illustrated that CRISPR-Cas9 might one day be useful for permanently eliminating genetic traits in the human species, but not without enormous risks. Despite the outcry that experiment drew, on April 8, 2016, another Chinese team published about their use of CRISPR to alter a gene in human beings that may be associated with susceptibility to the HIV infection.
The earlier Chinese experiment had already prompted calls for international regulation of CRISPR-Cas9 research. In December 2015, the International Summit on Human Gene Editing convened in Washington, DC, bringing scientists from around the world—including the Chinese teams, private sector researchers, and policy experts from the national security and ethics worlds—to the National Academy of Sciences to debate the future use of CRISPR-Cas9. In the lead-up to the summit, the White House weighed in, saying that “altering the human germline for clinical purposes is a line that should not be crossed at this time.” The U.S.-funded National Institutes of Health followed the White House’s cue, forbidding federal funding of human germline editing. After three days of deliberations, the summit’s international board concluded that it was “irresponsible” to conduct germline editing until safety and ethical consensus could be established.
Of particular concern, and taboo, the board said, was the use of CRISPR-Cas9 for enhancement of human traits: making children smarter or more athletic. The summit’s conclusions have no bearing on law anywhere in the world, and scientists have collectively agreed to set voluntary boundaries on their work. But few are in place in any country, including the United States, which allows scientific teams to change human inheritance genes if the groups can find nongovernmental funding.
Origins of Species
The greater challenge is to evolution, overall. CRISPR-Cas9 makes it possible to permanently alter the inherited traits of species with relative ease and minimal expense. The natural selection principles laid out in the nineteenth century by Charles Darwin may no longer apply: It is not “nature” that selects what orchids or frogs survive, but mankind. In some cases, the changes may be by design—making genetically altered animals that give birth to more mutants, which may, in turn, reproduce sexually with “normal” animals, thereby altering the inheritance permanently. In other cases, the evolutionary impact may be a mistake: CRISPR experiments have shown error rates as high as 60 percent in higher animals, meaning the Cas9 enzyme chops up far more than just the targeted genes.
Many scientists working in academia and the private sector, heavily funded by venture capitalists, are chopping up plant genes, creating new forms of biota that may solve far more problems than the “old-fashioned” genetically modified seed stock now commercially distributed—and may be exempt from all current forms of regulation, even in the United States. And despite ethical concerns expressed by the Chinese Academy of Sciences, a mix of government and private biotech and agricultural companies are racing to create new life forms in China.
For about a year and a half, biologists have been using so-called “gene drives”—a souped-up approach to CRISPR that operates at hyper-speed. Many refined gene drives have been put in play, allowing humans to ensure that 100 percent of offspring in a particular species will carry a given trait. It’s been done, for example, with disease-carrying mosquitoes, creating mutants incapable of spreading malaria.
Craig Venter, head of a research institute in La Jolla, California, that bears his name, recently upped the ante considerably by using CRISPR-Cas9 to create a completely novel life form. Dubbed JCVI-syn3.0, the creation announced on March 24 is unlike anything that exists in nature: It is alive and can self-reproduce, passing its genes on in a totally new stream of evolution. Venter and his team set out to create the most streamlined, minimalist cellular organism possible, using CRISPR to slice and dice existing bacterial genomes repeatedly until they whittled “life” down to just 473 genes. It is, Venter says, “a brand new, artificial species,” that doubles its population size every three hours, contained within the walls of his laboratory.
A Swiss biologist, Martin Fussenegger, told Nature magazine that he’s adding in genes to the genomes of a range of species, just to see what happens. And research teams in at least eighty-three countries, involving more than thirty thousand labs and potentially hundreds of thousands of scientists and technicians, are doing just that—playing with genes, to see what happens.