Many uses of CRISPR: Scientists say it all

Smartphones, superglue, electric cars, video chat. When does the wonder of a new technology stop? When you become so accustomed to its presence that you no longer think about it? When something new and good comes? How was it before when you forgot?

Whatever the answer, gene-editing technology CRISPR has not yet reached that stage. Ten years after Jennifer Dudna and Emanuel Charpentier discovered their first CRISPR, it has remained at the center of ambitious scientific projects and complex ethical discussions. It creates new ways to explore and revive old studies. Organic chemists use it and so do other scientists: entomologists, cardiologists, oncologists, zoologists, botanists.

For these researchers, there are still some surprises. But the excitement of complete innovation has been replaced by open possibilities and ongoing projects. Here are some of them.

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

But while Professor X has an affinity with superpowered human mutants, Dr. Martin is part of the red and juicy kind. “We’ve always longed for mutants, because it lets us understand our effectiveness,” Dr. Martin said of his research, which focuses on plant genomes in hopes of finding ways to make food – especially tomatoes – healthier, stronger and longer. Chronic.

When CRISPR-Cas9 arrives, a colleague of Dr. Martin offers to make mutant tomatoes as a gift. He was a bit skeptical, but, he told her, “I want a tomato that doesn’t produce any chlorogenic acid,” a substance that is thought to be beneficial to health; Tomatoes without it have never been found before. Dr. Martin wanted to remove what he believed was a gene sequence and wanted to see what happened. Soon a tomato without chlorogenic acid was in his lab.

Without looking for mutants, it was now possible to create them. “Finding these mutants, it was so efficient, and it was so wonderful, because it gave us confirmation of all these assumptions,” Dr. Martin said.

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

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

“I don’t claim that what we’ve done with vitamin D will solve any food insecurity problem,” said Dr. Martin, “but it’s just a good example. People like to get something they can stick to and it’s there. It’s No promises. ”

Infectious diseases

Christian Happy, a biologist who instructs the African Center of Excellence for the Genomics of Infectious Diseases in Nigeria, has spent his career identifying and capturing the spread of infectious diseases that spread from animals to humans. Many ways to do this are expensive and wrong.

For example, to perform a polymerase chain reaction or PCR test, you need to “go out to RNA, have a 60 60,000 machine, and hire someone specially trained,” Dr. Happy said. In most remote villages this type of test is expensive and logically impossible.

Recently, Dr. Happy and his colleagues used CRISPR-Cas13a technology (a close relative of CRISPR-Cas9) to detect diseases in the body by targeting genetic sequences related to pathogens. They were able to sequence the SARS-CoV-2 virus within weeks of the outbreak in Nigeria and were able to create a test that did not require any site equipment or trained technicians – just a tube to spit on.

“If you’re talking about the future of epidemic preparedness, you’re talking about that,” Dr. Happy said. “I want my grandmother to use it in her village.”

The CRISPR-based diagnostic test works well in heat, it is quite easy to use and costs one tenth of a standard PCR test. Nevertheless, Dr. Happy’s lab is constantly evaluating the accuracy of the technology and trying to persuade African public health leaders to adopt it.

He described their proposal as “cheap, fast, without the need for equipment and could be pushed to the far corners of the continent. It would allow Africa to occupy its natural place.”

Hereditary diseases

In the beginning was the zinc finger nucleus.

Gang Bao, a biochemical engineer at Rice University, was the first to use that gene-editing tool to treat sickle cell disease, an inherited disorder characterized by red blood cells. It took more than two years to develop Dr. Bao’s lab, and then the zinc finger nucleus would successfully cut the sickle cell sequence by about 10 percent of the time.

Another strategy took two more years and was only slightly more effective. And then, in 2013, shortly after using CRISPR to edit genes in living cells, Dr. Bao’s team changed the strategy again.

“To get some preliminary results from the beginning, CRISPR took us about a month,” Dr. Bao said. The method successfully spends about 60 percent of the time in the target sequence. 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 intentionally targeted? After multiple experiments on animals, Dr. Bao became convinced that the method would work for humans. In 2020 the Food and Drug Administration approved a clinical trial led by Dr. Matthew Portias and his lab at Stanford University, which is ongoing. And hopefully with the versatility of CRISPR, it can be used to treat other hereditary diseases. At the same time, other treatments that do not rely on gene editing have had success for 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 hopeful that a safe and effective gene-editing treatment for sickle cell will be available soon. How fast? “I think three to five more years,” he said with a smile.

Cardiology

It’s hard to change someone’s heart. And it’s not just because we’re often stubborn and stuck in our path. The heart produces new cells much slower than many other organs. Effective treatments in other parts of the human body are much more challenging with the heart.

It is difficult to know what is on someone’s mind. Even when you sequence a complete genome, there are often many segments that remain mysterious to scientists and doctors (called forms of uncertain meaning). A patient may have a heart condition, but there is no way to tie it to their genes. “You’re stuck,” said Dr. Joseph Wu, director of the Stanford Cardiovascular Institute. “So traditionally we just waited and told the patient we didn’t know what was going on.”

But over the past few years, Dr. Wu has used CRISPR to see what kind of effects the presence and absence of these confusing sequences have on heart cells, created in his lab from simulated pluripotent stem cell blood. By cutting specific genes and observing the effects, Dr. Wu and his colleagues were able to make connections between the DNA of individual patients and heart disease.

It will take a long time to treat these diseases with CRISPR, but diagnosis is a first step. “I think it’s going to have a big impact on personalized medicine,” said Dr. Wu, who noted that when he sequenced his genome, he found at least three forms of uncertain significance. “What do these variants mean to me?”

Sorghum is used in bread, alcohol and cereals all over the world. However it is not commercially made to the same degree as wheat or corn and, when processed, is often not tasty.

Karen Masell, a biotechnologist at the University of Queensland in Australia, saw a lot of room for improvement when she first began studying the plant in 2015. And because millions of people around the world eat sorghum, “if you make a small change, you have a huge impact,” he said.

He and his colleagues have used CRISPR to make Zerk frost tolerant, to make it heat tolerant, to prolong its growth period, to change its basic structure – “we use gene editing across the board,” he said.

Not only can this lead to tastier and healthier grains, but it can also make plants more resistant to changing climates, he said. But still editing crop genomes properly with CRISPR is no small task.

“We have no idea what the half-genes we drop do,” said Dr. Masel. “The second time we try to get in there and play God, we realize we’re a little bit away from our depths.” But, combined with more traditional breeding techniques using CRISPR, Dr. Masel is optimistic, despite being a self-described pessimist. And he hopes that further progress will lead to the commercialization of gene-edited foods, making them more accessible and more acceptable.

In 2012, a 6-year-old girl was suffering from acute lymphoblastic leukemia. Chemotherapy failed, and the case was too advanced for bone-marrow transplantation. There seems to be no other option, and the girl’s doctors told her parents to return home.

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

Since then, Dr. Carl June, a medical professor at the University of Pennsylvania who assisted in the development of CAR T-cell therapy, and his colleagues, including Dr. Ed Stadmawer, a hematologist-oncologist in pain medicine, have been working to improve it. This includes using CRISPR, the simplest and most accurate tool for editing T-cells outside the body. Dr. Stadmauer, who specializes in dealing with cancers of different blood and lymphatic systems, says that “the last decade or so has seen a revolution in the treatment of these diseases; It’s been fruitful and exciting. “

Over the past few years, Dr. Stadmauer has helped run a clinical trial where T-cells with significant CRISPR edits were inserted into patients with treatment-resistant cancer. The results were promising.

“Patients who had a very depressing prognosis are doing much better now, and some are recovering,” Dr. Stadmauer said. He continues to monitor patients, and sees that the performed T-cells are still present in the blood, ready to invade tumor cells in case of re-infection.

The real advantage is that scientists now know that treatment is possible with CRISPR.

“While it’s really science fiction-y biochemistry and science, the reality is that the field has moved tremendously,” says Dr. Stadmauer. He added that he was less excited about science than how useful CRISPR has become. “Every day I see 15 patients who need me,” he said. “That’s what inspires me.”

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