Kathy Krentz has played Mother Nature for most of her career, tinkering with the genomes of mice embryos before implanting the embryos in the wombs of recipient mother mice. After gestating for 20 days, the mutant litter is delivered to the researchers who ordered mice carrying a specific mutation.
“Mice make a good animal model because they reproduce very quickly, and you can get a cohort of animals that are genetically identical very quickly,” says Krentz, who runs UW-Madison’s Transgenic Animal Facility.
Aided by a powerful microscope, Krentz has injected hundreds of thousands of mice embryos with exogenous genes, inserted through a microscopic glass syringe. This process, known as transgenesis, was developed in the early 1970s. But it doesn’t always work.
Exogenous genes are expressed in offspring only if accepted by cells that store the biochemical units of heredity, known as the germline.
“The problem was that it would randomly insert DNA just anywhere on the genome,” she explains. “So as a cell divided, that piece of DNA would be incorporated into the dividing embryo, anywhere.”
In 2014, Krentz scrapped the laborious, error-prone process in favor of a new tool known as CRISPR-Cas9, despite her initial skepticism of the hype percolating around it.
“As we read scientific articles and looked at the data as to whether this was worth investing in, it became clear very quickly that CRISPR-Cas9 was going to be game changing,” she recalls. “And it has been.”
Andy Manis
Kathy Krentz with a mutant mouse: “It became clear very quickly that CRISPR-Cas9 was going to be game changing. And it has been.”
CRISPR-Cas9 is the biochemical equivalent of a magic wand. Compared to transgenics, CRISPR-Cas9 is a far faster and cheaper process. It also reaches targeted points on the genome with unrivaled precision. If the accuracy of transgenics was that of a free-fall bomb, CRISPR-Cas9 is a laser-guided missile, capable of editing.
UW-Madison researchers using CRISPR-Cas9 are circumspect about their ability to now make revisions to evolution’s handiwork over the last 3 or 4 billion years. But having been given the keys to the kingdom, so to speak, scientists around the globe are experimenting with their new creationist powers. It’s the end of the biological world as we know it.
“CRISPR has changed the mindset of science,” says Dustin Rubinstein, facility director of UW-Madison’s Biotechnology Center. “A lot of times technology is very straightforward. But with this, we’re only limited by our own creativity.”
The technology has already pointed toward some amazing possibilities. Scientists in London are working on eliminating the species of mosquitos that carry malaria. And the U.S. Food and Drug Administration last year approved for consumption Atlantic salmon that had been engineered to grow double the size of ordinary salmon in half the time. Experiments are being done on pig organs to turn off the genes that prevent cross-species organ transplants, which could eliminate organ-donor waiting lists.
Perhaps not since Gutenberg’s printing press have humans entrusted themselves with a tool so powerful, cheap, and time-saving; this may well be a watershed moment in human history — the world before CRISPR-Cas9 and the world after.
The ethical issues CRISPR-Cas9 raises don’t differ much from those raised by genetic engineering in general. The difference now is the speed at which these issues will arise. If scientific research was once a long, incremental slog, CRISPR-Cas9 has broken the sluices of discovery wide open.
“As it becomes cheaper and more accessible and more available, it’s going to expand the range of people who can pursue this kind of research and for more trivial purposes,” says Robert Streiffer, an associate professor of bioethics and philosophy at UW-Madison. “We’re not going to be able to put a lid on it as much as people might like.”
CRISPR is an acquired immunity found in bacteria, which evolved to fend off predatory viruses.
Because viruses are acellular, they reproduce by hijacking the reproductive machinery of host cells, provoking an immune response from the host. In humans, white blood cells are a first line of defense, but in bacteria it’s enzymes.
These viral-fighting enzymes are like an army defending the homeland from foreign foes, only its soldiers are weak and therefore victorious far less than 50 percent of the time. When the enzymes kill the foreign invaders, a highly specialized enzyme, Cas9, arrives on the battlefield to retrieve a piece of the snuffed out virus’ DNA.
Cas9 then returns to the bacteria’s genome and stuffs the piece of viral DNA in the CRISPR region of the genome. CRISPRs — an acronym for Clustered Regularly Interspaced Short Palindromic Repeats — are segments of repeating base pair sequences. The space between each repeat contains a piece of DNA from a virus defeated by the bacteria’s enzyme army and retrieved by Cas9.
The viral DNA stored between repeats then serves as a molecular wanted poster used by Cas9 as it patrols the interior of the bacterial cell reading the genomes of invading pathogens. If it encounters a sequence matching any of those on the wanted poster, Cas9 shreds the virus with its molecular scissors.
Seven years after its discovery, researchers at three different institutions independently have begun unlocking CRISPR-Cas9’s potential. By retooling its cellular mechanics, researchers soon turned CRISPR-Cas9 from a defensive system in bacteria into an offensive one in mammals and other organisms. They learned to rewire CRISPR-Cas9 cellular circuitry to make precise cuts anywhere along the genome of any organism, including humans.
In bacteria, Cas9 is dispatched from CRISPR regions of the genome; in animals — Krentz’s mice for example — CRISPR is a delivery device for Cas9, guided to its target by specially engineered molecules.
“Because the genome sequence has been identified, you find the sequence you want to edit and you can synthesize DNA that mimics that exact sequence,” Krentz explains. “Cas9 cuts the genome sequence exactly where we need it to be cut.”
Following its set of chemical instructions, it will delete, replace or add genes to the genome.
Rubinstein, known on campus as “the CRISPR guy,” performs the biochemical tweaks to CRISPR-Cas9 for Krentz and other researchers. At present, Cas9 is the gold standard among known CRISPR-associated enzymes. But he adds that biotech firms are screening bacteria in search of other enzymes that work differently or even better.
Known on campus as the “CRISPR guy,” Dustin Rubinstein performs the biochemical tweaks that allow other researchers on campus to alter genetic code.
“As this technology grows, we’ll discover more CRISPR-associated enzymes, ones more specific so you don’t have to worry about making changes where you don’t want, ones that make the changes more rapidly,” he says. “As we understand this more, we will fill the toolbox with more tools.”
Researchers are currently using this new tool to identify and tweak genes in plants and humans to do everything from preventing diseases to making improvements in crop quality.
And while we have known for some time how genes are ordered — genomes have been sequenced in thousands of organisms, including humans, since the 1990s — we don’t always know what each gene does or how they interact with one another.
Before CRISPR-Cas9, researchers like UW-Madison biochemist Richard Amasino learned the function of individual genes by blasting an organism — plants, in his case — with mutagenic chemicals or X-rays and then observing what traits were different or unexpressed.
“But there’s no way to do it without hitting genes all over the place,” he says. “But you go in with CRISPR and you have a very clearly defined plant at the other end. You know exactly what has been changed; it’s faster and more readily done.”
Amasino’s research focuses on how plants measure seasonal cues like sunlight and temperature to coordinate flowering. Once the function of enough genes are identified, the job descriptions for the remaining genes will be written even faster.
“It’s like a puzzle,” he says. “You start out turning over the pieces one-by-one, but at some point you’ve turned over enough of them to know you need a piece that looks a certain way because it has to fit over there.”
UW-Madison anthropology professor John Hawks expects the first widespread use of CRISPR-Cas9 in humans will be in the development of new gene therapies targeting genetic conditions like Huntington’s disease, a degenerative condition affecting the brain. Hawks says there are thousands of such conditions caused by a single gene mutation, but treatments are rare since, in many cases, sufferers worldwide number less than 100, he says.
With CRISPR-Cas9 the genes responsible for these conditions can be deleted and replaced in affected embryos with a fully functional version.
“When you know the cause and can potentially splice in a working version of the gene on someone already suffering from the condition, that kind of potential is very exciting,” Hawks says.
There are limits to what can be done, however.
“For things like Down syndrome, and even autism, I think the utility of CRISPR would be pretty low,” Hawks says. “There’s a developmental process that begins well before birth, and the downstream effects are in part based in the anatomy, not just metabolism. It’s not like something like sickle cell, where turning off the abnormal protein provides an effective therapy or even cure.”
Environmental pressures, genetic divergence and natural selection dating back to the single-celled organism from which all organisms descended have resulted in the human genome as it is today.
CRISPR-Cas9, Rubinstein says, places the levers of evolution into the calculated hands of people.
“It’s much more proactive,” he says. “Rather than saying, ‘We need more strains of bananas,’ now we can ask, ‘What can we do to improve bananas.’ It empowers us to take the initiative on certain global issues.”
About 90,000 years after modern humans emerged, they began planting seeds, not because it suddenly occurred to them to do so, but because localized climate change necessitated new survival strategies. Agriculture, which emerged independently and around the same time, in 11 places around the world, was possible because it fell within the genetically permissible range of behaviors unique to our species.
Corn was among the first crops domesticated, but, 10,000 years later, there is still a lot we don’t know about corn or the 35,000 genes packed onto its 11 chromosomes.
UW-Madison agronomy professor Shawn Kaeppler is the son of a Wisconsin dairy farmer who has spent his career studying corn genetics.
Kaeppler’s research is aimed at identifying the genes that influence corn’s resistance to disease, insects and pests. And in the context of climate change, he is exploring what genes might lead to a cultivar — a variety produced by selective breeding — that has better resistance to drought and heat or can develop root systems more tolerant of nutrient-poor soil.
“How can we make cultivars adaptable for not what Wisconsin is like now,” he explains, “but what it might be like 20 years from now.”
Sevie Kenyon
Shawn Kaeppler’s research attempts to identify the genes that influence corn’s resistance to disease and pests.
As with Amasino’s flowering research, CRISPR-Cas9 has accelerated Kaeppler’s research by making the process of gene identification faster and more precise. Already, he says, the gene-editing tool has led to wheat strains that are invulnerable to fungi and produce hardier crop yields.
Last month, the world’s largest bio-agriculture company, Monsanto, began researching how CRISPR-Cas9 might aid the engineering of seeds to meet specific environmental conditions. The move already is facing controversy, since CRISPR-Cas9 will allow Monsanto to sidestep federal regulations over genetically modified organisms.
Kaeppler says the difference between an organism that has been genetically modified and one genetically edited is largely semantics.
“Anything that is different from the thing you started with is in some way genetically modified,” he says.
Streiffer agrees, but explains that from a regulatory perspective, the government is concerned only with modifications where plant pathogens, like the cauliflower mosaic virus, are introduced into the recipient crop’s genome. CRISPR-Cas9 avoids these regulatory triggers because it doesn’t introduce foreign DNA or plant pathogens into the recipient genome.
Kaeppler says technology of any kind is neither good nor bad. He doesn’t see any concerns with using CRISPR-Cas9 to genetically modify consumer crop seeds.
“When you’re setting out to develop a better product and better modify things, I don’t have substantial concerns,” he says. “If you’re trying to do a good thing, it is highly improbable that you’ll get a negative result.”
Streiffer disagrees. He says genetically editing crops can raise a multitude of ethical issues. Modified crops can be a positive thing, if, for example, the yield is increased without having to increase the acreage or nutrients needed to grow it.
But the ethical implications can be far-reaching, he says.
“Maybe you get more profits for those farms, or less profits for farmers that don’t adopt that corn,” he says. “Is it really helping? Or is it just turning some people into winners and others into losers? Then you have to question why we’re investing so many resources into developing these kinds of things.”
Genetically modified crops have attracted farmers for a variety of reasons, but without much benefit for consumers.
“There was a lot of hype put into the existing genetically engineered crops on the market, and consumers eat them, basically, because they’re there,” he says. “But we don’t search them out because they’re especially attractive and especially good.”
Streiffer would like to see companies like Monsanto engineer crops with consumers in mind. Using CRISPR technology to produce fruit that has a longer shelf life is one idea.
“That would be wonderful,” he says.
There are similar hopes and concerns when it comes to gene manipulation in humans.
Could one day researchers master the genome so that children won’t throw tantrums? Not anytime soon, Hawks says.
He believes it will be a very long time before couples have the option of using CRISPR to select traits in their offspring since “we don’t know which genes make a difference.”
“What if someone wanted to design a super-intelligent human?” he asks. “We don’t know the first place to start.”
Human embryos have already been edited using CRISPR-Cas9, both in China and Sweden, with limited success. Aside from the mysteries around the complex interplay among genes, CRISPR isn’t 100 percent error-proof. Off-target mutations may not only throw a genome’s biochemical output out of whack, but also risks alterations to the germline, passing mutations onto subsequent generations.
“Say you go in and inadvertently mutate a tumor suppressor,” says Kris Saha, a UW-Madison molecular biology professor. “That’s a very troublesome scenario because now...you pass that mutation on to your kids.”
The appropriateness of modifications to an animal’s genome, Streiffer says, depends on whether it is good or bad for its welfare.
“We have examples of breeding where the interests of the animal itself hasn’t really been considered,” he says. “We have breeds of dog, for example, who have a very poor quality of life, but owners like them and they like them to be purebred even though they have various health issues.”
The GloFish became the first genetically modified animal on the consumer market four years after a researcher in Singapore gave a zebrafish the gene from a jellyfish that allowed it to glow fluorescent green.
Today, GloFish are bred in a variety of eclectic colors and appear to live happy lives.
“There are examples where a single gene has a pretty big effect,” says Kaeppler. “We can make a mutation to make corn have 30 to 40 stalks so it looks substantially different from what its progenitor looks like.”
Robert Streiffer, a bioethics and philosophy professor: “We’re not going to be able to put a lid on it.”
Streiffer believes CRISPR-related gene therapies and medical treatments may aggravate existing problems around equitable access, especially if one day rich couples can afford to design a child by making genetic revisions during embryonic development, with the traits in the offspring of poor couples left to chance.
“Issues of social justice will have to be addressed so that they don’t only benefit the wealthy, who already have a tremendous array of advantages,” he says. “There is a concern these things are going to exacerbate social and economic inequality.”
Among the certainties of CRISPR-Cas9 is that those with certain genetic conditions will one day be cured. In hemophiliacs, mostly men who’ve inherited a nonfunctioning gene on their X chromosome, CRISPR-Cas9 could edit out that gene, replacing it with a new one.
“Imagine if with CRISPR we could create normally functioning isolete cells, inject them into Type 1 diabetics and cure diabetes? That sort of thing is where you can see a huge difference being made if you give people more control over how cells work.”
One genetic disorder Hawks believes may be promising for CRISPR-related therapy is PKU, an enzyme deficiency that leads to mental retardation in children.
“It can be ameliorated with a diet that lacks a certain amino acid, but there is no cure,” he says. “Providing the ability to produce the necessary enzyme for infants and young children would effectively remove this as a disorder.”
At the dawn of the transgenics age, researchers not only knew what DNA was, but they could do stuff with it — cut it, stitch back together, implant it. But that doesn’t mean the public liked it.
According to a Pew Research Center survey in June 2016, a majority of Americans believe that gene-editing will “change society” and “negatives will outweigh the positives.” From Super Mice in the 1980s and FrankenFoods in the 1990s, to the completion of human genome sequencing in the 2000s and CRISPR-Cas9 today, the public has always been apprehensive about scientists “playing God.”
“We’ve gone through these iterations before in biology where we make this powerful new technique and people got concerned,” Rubinstein says. “With transgenics, everyone sat down and came up with a set of guidelines.”
That may not be possible with CRISPR-Cas9.
Rubinstein says with so many more labs across the globe, it wouldn’t be hard for poorly funded labs or rogue geneticists to cross ethical lines.
“It’s the slippery slope idea,” he says. “Is left-handedness something that needs to be cured? I don’t have any specific concerns.... The concern is more what we don’t know.”
