The many uses of CRISPR: Scientists tell it all

Smartphones, gum, electric motors, video chat. When does the wonder of a new technology take off? When you get so used to his presence that you no longer think about it? When something new and better comes? When you forget how things used to be?

Whatever the answer, CRISPR no-editing technology has not yet reached that point. Ten years after Jennifer Doudna and Emmanuelle Charpentier first unveiled their discovery of CRISPR, it has remained at the center of ambitious scientific projects and complex ethical discussions. It continues to create new avenues for exploration and revive 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 But the excitement of total novelty has been replaced by open possibilities and ongoing projects. Here are some 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 super-driven human mutants, dr. Martin partly of the red and juicy type. “We have always longed for mutants because it has enabled us to understand functionality,” said Dr. Martin said about her research, which focuses on plant genomes in the hope of finding ways to make food – especially tomatoes in her case – healthier, more robust and longer. lasting.

When CRISPR-Cas9 was added, one of Dr. Martin’s colleagues offered to make her a mutant tomato as a gift. She was somewhat skeptical, but, she told him, “I would rather have a tomato that produces no chlorogenic acid,” a substance that presumably has health benefits; tomatoes without it had not been found before. Dr. Martin wanted to remove what she believes was the key gene sequence and see what happened. Before long, a tomato was free of chlorogenic acid in her laboratory.

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

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

Dr. Martin explained that CRISPR can 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 do not claim that what we have done with vitamin D will solve any food insecurity problems,” said Dr. “But it’s just a good example. People like to have something they can hold on to, and it’s there. It’s not a promise.”

Communicable disease

Christian Happi, a biologist who runs the African Center for Infectious Disease Genomics in Nigeria, has devoted his career to developing methods to detect and limit the spread of infectious diseases that spread from animals to humans. . Many of the existing ways of doing this are expensive and inaccurate.

For example, to perform a polymerase chain reaction, or PCR, test, you have to “go to extract RNA, have a machine that is $ 60,000, and hire someone who is specially trained,” he said. Happi said. It is both expensive and logistically unlikely to take this type of testing to most remote villages.

Recently, Dr Happi and his collaborators used CRISPR-Cas13a technology (a close relative of CRISPR-Cas9) to detect diseases in the body by targeting genetic sequences associated with pathogens. They were able to follow up the SARS-CoV-2 virus within a few weeks of the arrival of the pandemic in Nigeria and develop a test that required no equipment on site or trained technicians – just a tube for spitting.

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

The CRISPR-based diagnostic test works well in the heat, is fairly easy to use and costs a tenth of a standard PCR test. Dr. However, Happi’s laboratory is constantly assessing the accuracy of the technology and is trying to persuade leaders in Africa’s public health systems to accept it.

He called their proposal one that is “cheaper, faster, that does not require equipment and can be printed in the farthest corners of the continent. It will allow Africa to occupy what I call its natural space. ”

Hereditary disease

In the beginning there was zinc finger nuclease.

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

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

“From the beginning to some initial results, CRISPR took us like a month,” said dr. Bao said. The method successfully cut 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 CRISPR affect genes that were not purposefully targeted? After a series of experiments in animals, Dr. Bao was convinced that the 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, which is underway. And there is also hope that it can be used with CRISPR’s versatility to treat other hereditary diseases. At the same time, other treatments that did not rely on gene editing were successful for sickle cell.

Dr. Bao and his laboratory 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 cell will be available soon. How soon? “I think another three to five years,” he said, smiling.

Cardiology

It’s hard to change someone’s heart. And it’s not just because we’re often stubborn and trapped 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 difficult to know what is in someone’s heart. Even when you have an entire genome sequence, there are often a number of segments that remain mysterious to scientists and doctors (called variants of uncertain meaning). A patient may have a heart condition, but there is no way to definitively bind it back to their genes. “You’re stuck,” said Dr. Joseph Wu, director of the Stanford Cardiovascular Institute, said. “So traditionally we would just wait and tell the patient we do not know what is going on.”

But over the past few years, dr. Wu uses CRISPR to see what effect the presence and absence of these confusing sequences has on heart cells, simulated in his laboratory with induced pluripotent stem cells generated from the blood. By excising specific genes and observing the effects, Dr. Wu and his collaborators were 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 it’s going to have a big impact in terms of personal medicine,” says Dr Wu, who mentioned that he found at least three variants of uncertain meaning when he got his own genome sequence. “What do these variants mean to me?”

Sorghum is used in bread, alcohol and cereals all over the world. But it is not commercially produced to the same extent as wheat or maize, and when processed, it is often not as tasty.

Karen Massel, a biotechnologist at the University of Queensland in Australia, saw a lot of room for improvement when she first started studying the plant in 2015. And because millions of people worldwide eat sorghum, “if you make a small change you can have a big impact,” she said.

She and her colleagues used CRISPR to make sorghum frost tolerant, to make it heat tolerant, to prolong its growth period, to change its root structure – “we use no editing in general,” she said.

Not only can this lead to tastier and healthier grains, but it can also make the 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 of the genes we knock out, we just have no idea what they do,” said Dr. Masel said. “The second we try to get in there and play God, we realize we’re a little out of our depth.” However, using CRISPR combined with more traditional breeding techniques, Dr. Massel is optimistic, despite being a self-described pessimist. And she hopes that further progress will lead to the commercialization of no-edited foods, making it 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 were apparently no other options, and the girl’s doctors told her parents to go back home.

Instead, they went to Philadelphia Children’s Hospital, where doctors used an experimental treatment called chimeric antigen receptor (CAR) T-cell therapy to stop the girl’s white blood cells from 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 his associates, including Dr. Ed Stadtmauer, a hematologist-oncologist at Penn Medicine, worked to improve it. This includes the use of CRISPR, which is the simplest and most accurate tool to modify T cells outside the body. Dr Stadtmauer, who specializes in the treatment of various types of blood and lymphatic system cancer, said that “over the past decade or so, there has been a revolution in the treatment of these diseases; it was rewarding and exciting. ”

Over the past few years, dr. Stadtmauer helped run a clinical trial in which T cells that underwent significant CRISPR editing were inserted into patients with treatment-resistant cancers. The results were promising.

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

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

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