The Many Uses of CRISPR: Scientists Tell All

Smartphones, superglue, electric cars, video chat. When does the new technology wear off? When do you get used to its presence that you don’t think of it anymore? When something newer and better comes along? When did you forget how things were before?

Whatever the answer, the gene-editing technology CRISPR has not reached that point yet. Ten years after Jennifer Doudna and Emmanuelle Charpentier first introduced their discovery of CRISPR, it remained at the center of ambitious scientific projects and complex ethical discussions. It continues to create new avenues for exploration and reinvigorate old studies. Biochemists use it, and so do other scientists: entomologists, cardiologists, oncologists, zoologists, botanists.

For these researchers, some of the wonder is still there. But the total novelty of the excitement has been replaced by open possibilities and ongoing projects. Here are a few of them.

Cathie Martin, a botanist at the John Innes Center in Norwich, England, and Charles Xavier, founder of the X-Men superhero team: They both love mutants.

But while Professor X has an affinity for superpowered human mutants, Dr. Martin is partial to the red and juicy type. “We always craved mutants, because that allowed us to understand functionality,” Dr. Martin said of her research, which focuses on plant genomes in hopes of finding ways to make foods – especially in tomatoes her case – healthier, more robust and longer lasting.

When CRISPR-Cas9 came along, one of Dr. Martin’s colleagues offered to make her a mutant tomato as a gift. She was slightly skeptical, but, she told him, “I would quite like a tomato that produces no chlorogenic acid,” a substance thought to have health benefits; without tomatoes it had not been found before. Dr. Martin wanted to remove what she believed was the key gene sequence and see what happened. Soon a tomato without chlorogenic acid was in her lab.

Instead of looking for mutants, it was now possible to create them. “Getting those mutants, it was so efficient, and it was so wonderful, because it gave us confirmation of all these hypotheses we had,” Dr. Martin said.

Most recently, researchers at Dr. Martin’s lab used CRISPR to create a tomato plant that can accumulate vitamin D when exposed to sunlight. Just one gram of the leaves contains 60 times the recommended daily value for adults.

Dr. Martin explained that CRISPR could be used across a broad spectrum of food modifications. It could potentially remove allergens from nuts and create plants that use water more efficiently.

“I don’t claim that what we did with vitamin D will solve any food insecurity problems,” Dr. Martin said, “But it’s just a good example. People like to have something that they can hang on to, and this is there. It’s not a promise. “

Infectious Disease

Christian Happi, a biologist who directs the African Center for Excellence in Genomics of Infectious Diseases in Nigeria, has spent his career developing methods to detect and prevent the spread of infectious diseases that spread from humans to animals. Many of the existing ways to do so are costly and inaccurate.

For example, in order to perform a polymerase chain reaction, or PCR, test, you need “to go extract RNA, have a machine that is $ 60,000 and hire someone who is specially trained,” Dr. Happi said. It’s both costly and logistically implausible to take this kind of testing to the most remote villages.

Recently, Dr. Happi and its collaborators used CRISPR-Cas13a technology (a close relative of CRISPR-Cas9) to detect pathogens associated with body-targeting genetic sequences in patients. They were able to sequence within the SARS-CoV-2 virus within a couple of weeks of pandemic arrivals in Nigeria and develop a test that requires no on-site equipment or trained technicians – just a tube for spit.

“If you ‘re talking about the future of pandemic preparedness, that’ s what you ‘re talking about,” Dr. Happi said. “I want to use my grandmother in this village.”

The CRISPR-based diagnostic test functions well in the heat, is quite easy to use and costs one-third of a standard PCR test. Still, Dr. Happi’s lab is continually assessing the accuracy of technology and trying to persuade leaders in the African public health systems to embrace it.

He called one of their proposal “that is cheaper, faster, that does not require equipment and can be pushed into the remotest corners of the continent. This would allow Africa to occupy what I call its natural space. “

Hereditary Illness

In the beginning there was a zinc finger nuclease.

That was the gene-editing tool that Gang Bao, a biochemical engineer at Rice University, used to first try to treat sickle cell disease, a inherited disorder marked by red blood cells. It took Dr. Bao’s lab has more than two years of development, and then zinc finger nuclease will successfully cut the sickle cell sequence by only about 10 percent of the time.

Another technique took another two years and was only slightly more effective. And then, in 2013, soon after CRISPR was used to successfully edit genes in living cells, Dr. Bao’s team changed tack again.

“From the beginning to having some preliminary results, CRISPR took us like a month,” Dr. Bao said. This method successfully cuts the target sequence about 60 percent of the time. It was easier to make and more effective. “It was just amazing,” he said.

The next challenge was to determine the side effects of the process. That is, how did the CRISPR affect genes that weren’t being purposefully targeted? After a series of experiments in animals, Dr. Bao was convinced that this method would work for humans. In 2020 the Food and Drug Administration approved a clinical trial, led by Dr. Matthew Porteus and his lab at Stanford University, that is ongoing. And there is also hope that with CRISPR’s versatility, it may be used to treat other hereditary diseases. At the same time, other treatments that have not relied on gene editing have had a success for the sickle cell.

Dr. Bao and his lab are still trying to determine all the secondary and tertiary effects of using CRISPR. But Dr. Bao is optimistic that a safe and effective gene-editing treatment for sickle cells will be available soon. How soon? “I think another three to five years,” he said, smiling.

Cardiology

It is hard to change someone’s heart. And that’s not just because we are often stubborn and stuck in our ways. The heart generates new cells at a much slower rate than many other organs. Treatments that are effective in other parts of the human anatomy are much more challenging with the heart.

It is also hard to know what is in one’s heart. Even when you sequence an entire genome, there are often a number of segments that remain mysterious to scientists and doctors (called variants of uncertainty). A patient may have a heart condition, but there is no way to tie it definitively back to their genes. “You’re stuck,” said Dr. Joseph Wu, Director of the Stanford Cardiovascular Institute. “So traditionally we would just wait and tell the patient if we didn’t know what was going on.”

But over the past couple of years, Dr. Wu has been using CRISPR to see what kind of effects the presence and absence of these befuddling sequences have on heart cells, simulated in his lab-induced pluripotent stem cells generated from the blood. By cutting out particular genes and observing the effects, Dr. Wu and his collaborators have been able to draw links between the DNA of individual patients and heart disease.

It will be a long time before these diseases can be treated with CRISPR, but diagnosis is a first step. “I think this is going to have a big impact in terms of personalized medicine,” said Dr. Wu, who mentioned that he found at least three variants of uncertain significance when he got his own genome sequenced. “What do these variants mean for me?”

Sorghum is used in bread, alcohol and cereal all over the world. But it has not been commercially engineered to the same degree as wheat or corn, and, when processed, it is often not as tasty.

Karen Massel, a biotechnologist at the University of Queensland in Australia, saw quite a bit of room for improvement when she first started planting in 2015. And because millions of people eat sorghum worldwide, “if you can make a small change you can A huge impact, “she said.

She and her colleagues have used CRISPR to try to make sorghum frost tolerant, to make it heat tolerant, to prolong its growth period, to change its root structure – “we use gene editing across the board,” she said.

Not only could this lead to more delicious and healthier cereals, but it could also make plants more resistant to the changing climate, she said. But it is still no small task to accurately edit the genomes of crops with CRISPR.

“Half the genes that we knock out, we just have no idea what they do,” Dr. Massel said. “The second we try to get in there and play God, we realize we’re a bit out of our depth.” But, using CRISPR combined with more traditional breeding techniques, Massel is optimistic, despite being a self-described pessimist. And she hopes that further advances will lead to generating commercialized gene-edited foods, making them more accessible and more acceptable.

In 2012, a 6-year-old girl suffered from acute lymphoblastic leukemia. Chemotherapy was unsuccessful, and the case was too advanced for a bone-marrow transplant. There didn’t seem to be any other options, and the girl’s physicians told her parents to go back home.

Instead, they went to the Children’s Hospital of Philadelphia, where doctors used an experimental treatment called chimeric antigen receptor (CAR) T-cell therapy to turn the girl’s white blood cells against the cancer. Ten years later, the girl is cancer free.

Since then, Dr. Carl June, a medical professor at the University of Pennsylvania who helped develop CAR T-cell therapy, and its collaborators, including Dr. Ed Stadtmauer, a hematologist-oncologist at Penn Medicine, has been working to improve it. That includes using CRISPR, which is the simplest and most accurate tool to edit T-cells outside the body. Dr. Stadtmauer, who specializes in dealing with various types of blood and lymph system cancers, said that “the last decade or so has just seen a revolution treating these diseases; It has been rewarding and exciting. “

Over the past couple of years, Dr. Stadtmauer helped run a clinical trial in which T-cells that underwent significant CRISPR editing were included in patients with treatment-resistant cancers. The results were promising.

“Patients that had very dismal prognoses are doing much better now, and some are being cured,” Dr. Stadtmauer said. He has continued to monitor the patients, and has found that the edited T-cells are still present in the blood, ready to attack tumor cells in a relapse of the case.

The real benefit is that scientists now know that CRISPR-aided treatments are possible.

“Even though it’s really sort of science fiction-y biochemistry and science, the reality is that the field has moved tremendously,” Dr. Stadtmauer said. He added that he was less excited by the science than how useful CRISPR had become. “Every day I see maybe 15 patients who need me,” he said. “That’s what motivates me.”

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