August 21, 2014

New technique accelerates genome editing process

It sounds like a potato chip. But CRISPR is actually the acronym for a new genome editing technique that, by many accounts, is accelerating the study of genes and disease.

It sounds like a potato chip.

But CRISPR is actually the acronym for a new genome editing technique that, by many accounts, is accelerating the study of genes and disease.

An artist’s depiction shows the CRISPR-Cas9 genome editing technique. (Courtesy of Stephen Dixon and Feng Zhang, MIT)

“It is a truly revolutionary technology that will greatly change life sciences and medicine,” predicted Wenbiao Chen, Ph.D., who in collaboration with Susan Wente, Ph.D., and Wente’s former postdoctoral fellow, Li-En Jao, Ph.D., described a high-efficiency CRISPR technique in zebrafish last summer in the Proceedings of the National Academy of Sciences.

Jao recently joined the faculty at the University of California, Davis. Hundreds of labs around the world have now adopted their specific zebrafish technology, Chen said.

With Vanderbilt colleague Richard O’Brien, Ph.D., Chen is using CRISPR to identify genetic mutations that contribute to insulin resistance and beta cell dysfunction in type 2 diabetes. Their work could lead to new treatments for the disease, which is at epidemic proportions in this country.

CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, was first described two years ago. It’s an adaptive immune response bacteria use to recognize and thwart “invasions” of viral and foreign DNA.

“It turns out that some species of bacteria have an ingenious system to protect themselves from viruses,” said Douglas Mortlock, Ph.D., scientific co-director of the Vanderbilt Transgenic Mouse/Embryonic Stem Cell Shared Resource.

“If viral DNA gets into their cell, they cut off a tiny bit of it and stick it in their own chromosome and later they can make an RNA copy of it … to scan any new invading DNA sequences,” he said. “If they match, it cuts the DNA.”

About a half dozen Vanderbilt labs are now using the technique, and more are ramping up to do it, he said.

A common way to study a mutation thought to cause human disease is to mutate the normal gene in an animal model, like the mouse, and see what happens.

Other gene mutation methods in mice can take 18 months and cost up to $20,000. “Now we can basically squirt this stuff into mouse embryos and three weeks later mice are born that have the mutation … at a cost of $3,000 or less,” Mortlock said. “It’s stunning.”

This “stuff” is CRISPR, a piece of the RNA copy of the gene that is being targeted, and Cas9, an enzyme that can cut both helical strands of DNA. When injected into the cell, the RNA “guides” Cas9 to the gene so it can be snipped and disabled or “knocked out.”

If a piece of DNA is also co-injected, new genetic material can be “sewn into” the cell’s genetic material.

Before this can be applied to treating actual patients, however, researchers will need to determine the specificity of the technique. Does it actually “fix” the defective gene that is being targeted, without changing the genetic code elsewhere in the genome, in other words, without causing unforeseen “off-target” consequences?

That said, Vanderbilt researchers are using CRISPR to accelerate studies involving human “induced pluripotent” stem or iPS cells. These are skin cells that have been converted stem cells, and then “reprogrammed” to become neurons or other cell types.

“There is some reason to be optimistic that (CRISPR) will be a game changer for us,” said Kevin Ess, M.D., Ph.D., who has been making iPS cells to study tuberous sclerosis complex (TSC), one of the most common genetic causes of seizures and autism in children.

With CRISPR, Ess and his colleagues can introduce a mutation associated with TSC into a normal cell, or fix the mutated gene in a cell taken from a patient. The goal is to determine what the gene does, and how the mutation disrupts normal brain function.

“I think this is the beginning of true regenerative medicine,” Ess said. Not only is CRISPR accelerating the search for potential new drugs to treat TSC and other neurological disorders, but “maybe it will help us to fix your cells and give them back to you.”

Similarly, Aaron Bowman, Ph.D., and his colleagues are using CRISPR to evaluate the interactions between genetics and environment in neurological conditions including Huntington’s disease, Parkinson’s disease and Restless Legs Syndrome.

Charles Hong, M.D., Ph.D., and his colleagues use iPS cells to identify genes associated with as-yet-characterized heart conditions. “Maybe one day we can … correct the gene in a petri dish, and put the cells back.

“That’s probably 20 years away,” Hong said, but CRISPR, which he is currently bringing to his lab, could accelerate that timetable significantly.

“It is remarkably robust technology,” said Ian Macara, Ph.D., chair of the Department of Cell & Developmental Biology. “The first time we tried it, it worked … with amazing efficiency.”

Here’s the rub: “No translational research in the world, no matter how many billion dollars you spent on it, ever would have discovered CRISPR,” he said. “Never, because CRISPR came out of people studying obscure bacterial behaviors.

“It’s just pure scientific curiosity,” Macara said. “That’s the only driving force for studying this. How life works. That’s the wonderful thing about studying biology – the unexpected. It comes out of the blue. It is the coolest thing.”