Gene Editing

Although human life is robust, at times it can be fragile. For people with diseases like cystic fibrosis and sickle cell anemia, their disease is produced by a change in only one letter of DNA.

DNA is written with just four letters, called bases: A, T, G, and C. A small change, or mutation, can cause the DNA to build the wrong proteins in the body. Now, scientists have found a new way to edit these DNA instructions.

The team, located at the Gladstone Institutes, have combined existing technologies in a way that no one has before, with entirely new results.

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One in a Thousand

DNA isn’t hard to edit, but when a scientist tries to edit a batch of cells in the lab, only a few actually accept the changes. “The problem that we face is that when we edit DNA and change a single base in the genome of one cell, it’s by nature a rare event,” explained Bruce Conklin, Senior Investigator at the Gladstone Institutes. “It’s only one cell in a thousand.”

For most research purposes, this isn’t a problem. In addition to making the desired edit to the DNA, the scientist can also add a 300-base-long piece of DNA that makes it resistant to antibiotics. Then they dose their mutated cell cultures with antibiotics, killing off all the cells that resisted the edit. “The only ones that survive are the ones that have this marker,” said Conklin.

If a scientist is adding or subtracting entire genes, which can be hundreds or thousands of bases long, adding 300 extra bases doesn’t make much difference. But for single letter mutations, adding so many extra letters can change the way the DNA behaves.

“If you’re wanting to correct a genetic mutation, you don’t want to have to leave this DNA in there that was used as a marker to identify the cells,” Conklin said. “For practical purposes, that’s how we’ve made transgenic mice and everything else. But as we move toward wanting to correct or model human diseases, then there’s an increased desire to exactly replicate the disease or the healthy state, depending on what you’re studying.”

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Four Techniques, One Goal

“What we’ve done is just changed that one letter and tried to find a way to identify those cells without adding that extra paragraph,” said Conklin.

First, they used a genetic editing technique called TALENs to cut open the strand of DNA containing the section they want to edit. “The cuts are made in such a way that when the cells repair it, that one base is changed from the wrong letter that makes a person sick to the right letter that would make them better,” explained Conklin. The technique, however, only produces results in one cell in 1,000.

With the edits complete, the team then had to grow their new edit in living cells. They were particularly interested in induced pluripotent stem cells (iPS cells), which can be made from the mature cells of any person. “iPS cells have traditionally been very difficult and tedious to grow, but we were able to work out the culture conditions in such a way that they became much [easier] to grow,” Conklin said.

Next, they divided the cells into 96 different growth wells, with only 2,000 cells in each well, and let the cells grow and multiply. Then, using a technique called sib selection, they split off roughly 30 percent of each well’s cells for testing with a tool called droplet digital PCR.

Once they identified which growth wells had cells that had taken up their new mutation, they split apart the best well and seeded 96 new wells. Rather than 0.05 to 0.1 percent of the cells in each well with the mutation, as in the first round, about 1 percent of the cells in the second round carried the mutation. By the third round, 30 to 40 percent of the cells were mutants.

“Sometimes by the third round, we have an almost pure population,” Conklin said. “This has increased ten to a hundredfold our ability to make these single base changes.”

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A Golden Age of Gene Editing

Conklin is excited about the applications of their new method. “It’s been almost herculean to get a single base change like we’ve been doing routinely,” he said.

He hopes that this technique will soon be used to help treat, or even cure, genetic diseases. “It’s not that far off,” he said. “There are already clinical trials for using iPS cells for human transplants. If I were to have a genetic disease and someone were to make new tissue and give it back to me, I’d prefer that the genetic disease was corrected.”

For example, Conklin said, there is a genetic disease that causes blindness, and there are clinical trials now underway to take a blind patient's skin cells, turn them into iPS cells, and inject them into the retina of his or her eye to grow a new, healthy retina.

Using the Gladstone Institutes' technique, scientists could correct the genetic defect, so the new retina would be healthy and not degrade over time. Researchers believe the patient's body would not reject the new retina, since it's made from the patient's own cells.

Conklin admits that the process of changing the DNA code will never be simple. “It’s going to be very expensive and complicated. It’s not an easy process,” he said. But he remains optimistic.

“The four technologies [we used] are all improving exponentially," Conklin said. "You can plan on them getting better dramatically."

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