Scientists in China successfully cured 24 mice of their eye condition, which was produced by a single, mutant copy of one gene. That demonstration, reported in the scientific literature two years ago, was billed as the first to show that it’s possible to correct a genetic disease using a genome editing tool, which scientists call CRISPR-Cas9. Although in mice, the findings offered the first proof of principle that scientists and doctors might one day have sufficient skill and precision to edit single-gene disorders out of human genomes in much the same way.
Jinsong Li, one of the leaders of the study from the Chinese Academy of Sciences, said then that he believes it is “absolutely possible to use CRISPR to cure genetic disease in the near future.” As further evidence in support of Li’s conclusion, his paper came out alongside another by researchers in the Netherlands. They had used CRISPR to correct a gene that causes cystic fibrosis in adult stem cells derived from patients with the single-gene disorder.
Early last year, scientists welcomed the first monkeys, a pair of twins, carrying mutations that had been specifically edited into their genomes using CRISPR. A few months later, scientists announced that they’d used CRISPR to correct a genetic condition by editing the liver cells of mice — the first time the procedure was shown to work in an adult animal.
In a perspective piece published in the New England Journal of Medicine this year, Eric Lander, director of the Broad Institute, envisioned other therapeutic futures for CRISPR. The molecular tool set could be used to treat a progressive and genetically encoded form of blindness by inactivating a mutant gene in light-sensitive cells of the retina; it could prevent heart attacks in patients with a condition that leads to extremely high cholesterol by editing a gene in the liver; or it could edit sickle cell disease or hemophilia right out of the bloodstream. The immune cells of patients with HIV might be edited to resist the infection by eliminating the “door” that allows the virus to enter their cells.
There are important caveats and concerns when it comes to therapeutic uses of CRISPR, so read on, but the possibilities are seemingly endless. In March, researchers at the Salk Institute revealed another way that CRISPR might beat HIV infections. When HIV finds its way into human cells, the virus stages a takeover, inserting itself into the genome to convert human cells into HIV factories that produce more and more of the virus. That, after all, is what viruses do. In many cases, our immune systems do a good job of recognizing infections and mounting attacks against them. Unfortunately, our immune cells aren’t well equipped to recognize and fight off HIV.
Hsin-Kai Liao and Juan Carlos Izpisua Belmonte wondered if they could program CRISPR to seek out and chop up those viruses even in the places where they can hide, safely embedded in the human genome. The Salk researchers presented evidence in Nature Communications that they could indeed use CRISPR against HIV in human cells. In healthy cells carrying their version of CRISPR, HIV couldn’t set up shop in the first place.
The Salk team’s demonstration was inspired in part by the defensive work that CRISPR-Cas9 has been doing for millions of years before we humans discovered it and figured out how to use it for our own purposes. Bacteria, too, must contend with the regular threat of viral infection, and CRISPR is borrowed from them. It’s part of an adaptive immune system that allows bacteria to recognize and destroy key bits of viral DNA. By inserting snippets of viral DNA into their CRISPR genes, bacteria build a “memory” of infection, which serves to protect them against later attacks.
By 2012, Jennifer Doudna, of the University of California, Berkeley, and Emmanuelle Charpentier, of Sweden’s Umeå University, had realized that CRISPR-Cas9 — first described by others in the 1980s — wasn’t just a bacterial immune system. As they learned more about how it works in bacteria, they began to recognize the CRISPR system as a new and potentially more effective means for editing genomes. Cas9 is an enzyme that cuts DNA, and Doudna and Charpentier showed that CRISPR-Cas9 could be readily programmed to target and then cut any DNA sequence, simply by attaching it to the right guide.
“We can direct it to any site we select,” Doudna said in an announcement issued by the Howard Hughes Medical Institute at the time. “Because the guide RNA contains both the structure required for Cas9 binding and a separate guide sequence that can base pair with DNA, we can program Cas9 to cleave a specific site by simply changing the guide sequence.”
The system offered a new method for introducing new genetic information into cells in a very directed way. Doudna and Charpentier hadn’t yet shown that CRISPR worked inside cells, but they knew they’d hit on something that could change the future of genome editing. By the following year, it had become clear that CRISPR-Cas9 does work in cells, human cells included.
There are many ways to think about what CRISPR now makes possible. Sometimes CRISPR has been likened to programmable DNA scissors, but more often it’s described as a kind of Swiss Army knife, a reference to how versatile and handy it is for editing or otherwise manipulating essentially any gene. New versions of CRISPR-Cas9 include modifications to activate or suppress genes or gene combinations instead of cut them.
CRISPR-Cas9 is not the only form of CRISPR. Recently, Feng Zhang, a CRISPR pioneer at the Broad Institute of MIT and Harvard, conceived another version of the system by swapping out the Cas9 protein for a different protein, Cpf1. According to some, CRISPR-Cpf1 is a much simpler and more precise gene-editing tool than CRISPR-Cas9.
CRISPR isn’t the first or the only tool available for editing genomes in this way, but it is the most readily adaptable and the fastest. As George Church, another luminary in the field of genomics, has explained it on more than one occasion, “It’s like you throw a piston into a car and it finds its way to the right place and swaps out with one of the other pistons — while the motor’s running.”
Suhani Vora, a member of the Church lab at Harvard’s Wyss Institute for Biologically Inspired Engineering, says CRISPR is the plug-and-play system that geneticists have been looking for. To modify CRISPR in any way that suits them, researchers simply order a little piece of RNA that targets the system to go to any desired location in the genome. They can also easily attach other basic components, to grow the tool kit from basic genome editing to one including gene control or even imaging.
CRISPR is also cheap. While earlier genome editing tools have their advantages for certain applications, with the most comparable technology, it “would have taken $2 million and a year to build a library targeting every gene in the human genome,” Vora says. “Now, with CRISPR, we can build that same library in the span of a week, spending only a few thousand dollars.”
Compared to other genome-editing tools of molecular biology, which can often be finicky and difficult, she adds, CRISPR “just sort of works.” Considering what was possible before CRISPR engineering, Vora says, “it’s mind-blowing how much genetics we can do within a short span of time.”
As a result, the news on CRISPR is coming out at such a fast and furious pace these days that it’s a challenge just to keep up. As noted in a recent blog post by Jacob Corn, the scientific director of the Innovative Genomics Initiative at the University of California, Berkeley, new scientific papers related to CRISPR-Cas9 literally come out every day. If this sounds like good news only for those deeply invested in basic biological research, it isn’t.
As Boehm explains it, CRISPR is coming along at an “unprecedented moment” in the history of biomedicine. The Human Genome Project gave us the first complete (or nearly complete) map of the human genome. The success of that effort was quickly followed by a flood of information connecting particular genes and gene combinations to all sorts of complex and chronic health conditions: cancer, heart disease, diabetes, and obesity, among them.
“A decade ago we didn’t have the faintest idea which gene mutations caused most diseases,” Boehm says. “It would have been interesting to edit genes and turn them on and off, but there was no real urgency to do so because we didn’t have the map.”
Now, scientists have the map, along with lists of genes — some of them quite lengthy — that appear to be important for one disease state or another. But in many cases, they still don’t know what those genes really do or, in the case of more complicated conditions involving many genes, how those genes influence each other. Boehm points to the fact that cancer has been tied to perhaps 1,000 genes and probably 100,000 mutations.
“Now [with CRISPR] we can engineer thousands of mutations and study their consequences,” Boehm says. “That’s never before been possible.”
That’s exactly what the Broad’s collaborative team is busy doing with that long list of cancer-related genes. They are engineering mutations into cells with CRISPR and studying their consequences by injecting those cells back into mice to see whether they form tumors, for instance, or examining their activities at the molecular level “to understand cancer at a massive scale.” They’re also turning off every gene within cancerous cells to find all of the molecular ingredients that cancers depend upon for survival. Boehm says the effort will yield a road map for the pharmaceutical industry to guide researchers toward new kinds of targeted treatments.
One of the challenges for treatments that target particular cancer mutations is that tumors evolve and find ways to resist. The Broad team also plans to identify all of those “escape routes” for use in strategically assembling more effective or longer-lasting drug combinations. And cancer offers just one example of the progress to be made in medical research and treatment through the application of CRISPR, whether or not the genome editing technology ultimately finds its way into clinics or drugs directly.
Zhang, from the Broad Institute, says one way to think of it is that “CRISPR has taken basic research from analog to digital. Not only is CRISPR greatly accelerating the pace at which researchers can test theories about human health and disease, but it is leveling the playing field by allowing researchers around the world, working on all sorts of organisms, to precisely engineer genomes. There are thousands of talented scientists out there who are experts on various human diseases, and CRISPR is empowering them to rapidly create models that can be used to explore specific diseases and assess the impact of genetic variation on disease outcomes with a speed that wasn’t imaginable before.”
In other words, CRISPR has democratized genome engineering and genome editing by making it so easy and accessible that just about any molecular biology lab anywhere can do it. Zhang says he’s excited about what CRISPR can tell us “about how our bodies respond to drugs and how our immune systems function.” He notes that CRISPR can provide important insights into the brain and psychiatric diseases, which “have been notoriously difficult to disentangle.”
But CRISPR and genome engineering in general raise a series of bigger, more philosophical questions too. It might be just fine to correct a mutant gene in a mouse by injecting Cas9 and a single guide RNA into a developing zygote. In humans, most aren’t troubled by the thought of correcting a gene in a particular adult tissue, such as the eye or the liver. But many people do get very nervous about the possibility that CRISPR might be put to work in engineering humans before they are born in ways that would be passed on to the next generation.
Fundamental questions about the ethics of engineering humans aren’t specific to any one tool or another, but the ease of CRISPR has made human engineering seem that much more likely, even inevitable. Earlier this year scientists at China’s Sun Yat-Sen University reported that they had applied gene editing with CRISPR-Cas9 to modify a gene responsible for beta-thalassemia in human zygotes. Recognizing the ethical concerns of work in viable human embryos, the researchers chose to use embryos they knew to be incapable of developing into fetuses. Their findings also led them to conclude that use of CRISPR-Cas9 for editing human embryos “may be premature at this stage.”
Before the Chinese report made any headlines, experts led by Edward Lanphier, president and chief executive officer of Sangamo BioSciences in Richmond, California, and chairman of the Alliance for Regenerative Medicine in Washington, D.C., published a comment in Nature under the heading “Don’t edit the human germ line.” The comment, inspired by rumors that such efforts were already under way, noted “grave concerns” about ethics and safety and the fear that such efforts could have negative repercussions for therapeutic genome editing in the rest of the body.
In fact, Sangamo BioSciences has been working toward therapeutic uses for genome editing for years, using an earlier and more arduous technique based on zinc finger nucleases. Clinical trials of their zinc finger-based treatments are already being done in patients with HIV, with more trials to come in single-gene disorders including hemophilia, beta-thalessemia, Huntington’s, and sickle cell disease.
Many organizations and institutions are convening to help provide guidance on the ethical issues of gene editing. Based on a meeting held in Napa, California, in January 2014, a group including Church, Doudna, and many others called for a “prudent path forward” and “framework for open discourse on CRISPR.” The United Nations Educational, Scientific, and Cultural Organization’s International Bioethics Committee arrived at a more stringent mandate, calling for a moratorium on germ line editing of the human genome, citing concerns over introducing “hereditary modifications, which could be transmitted to future generations.”
In December, the International Summit on Human Gene Editing was held to discuss issues surrounding CRISPR. The organizing committee concluded that gene-editing research should continue in cells that would not pass to the next generation but that it would be “irresponsible to proceed” with germ line editing until the risks and benefits are clearly determined and the ethical and societal concerns resolved. An ongoing forum comprised of international scientific and medical societies will continue to discuss the progress of research.
When asked whether he thought there was a “right way” to apply this technology to erase diseases from the germ line — well before a baby is born — or whether that should always be off-limits, Lanphier turned the question back around. “If one assumes that you can perfect the science, perfect the technique — and I think one should assume that,” he said, then how should the technology be put to use? Is it acceptable only when treating a disease? What about genome editing to enhance or alter traits in a perfectly healthy person?
“That’s precisely the discussion that we as a global society need to have, because this is an incredibly powerful technology and for the first time makes it very approachable and — in a relatively short period of time — almost trivial from a technical perspective to do,” Lanphier said. “The question for us as a global society is: Should we do it?”