Building off the powerful gene-editing technology known as CRISPR, researchers have created a new tool that can target and change a single letter in a sequence of DNA.
The new technique, known as base editing, can be programmed to turn the DNA base cytosine (known in the DNA alphabet as C) into the base uracil, which acts like a thymine (known as T).
It may be a small substitution, but it can have a huge impact, experts say.
In a paper published Wednesday in Nature, researchers describe how they used base editing to correct a genetic variant associated with late-onset Alzheimer's disease as well as another variant associated with breast cancer.
"Most human genetic variants that cause disease are point mutations -- a single base pair that for whatever reason has been changed to a different base pair," said David Liu, a chemical biologist at Harvard University and the senior author of the paper. "So we set out to take a new approach to genome editing that would be especially good at correcting point mutations."
The science of genome editing has been revolutionized in the last few years thanks to the widespread use of CRISPR-Cas9, which was invented in 2012.
While the CRISPR system is excellent at finding and disrupting specific genes, it is not particularly good at fixing point mutations within them.
CRISPR consists of two parts: The first is a strand of RNA that scientists have programmed to locate a string of letters in a DNA sequence. The second is an enzyme -- usually Cas9 -- that serves as a pair of molecular scissors.
When the system is inserted into a cell, the RNA guides the Cas9 to a particular part of the genome. Then the enzyme makes a two-stranded break in DNA's double helix.
Traditionally, if researchers want to correct a point mutation within a gene, they would also deliver an additional piece of DNA to the cells. Because cells don't like having their DNA cut, they try to make repairs right away. The hope is that the cell will use the newly delivered DNA to fix the break, thereby allowing scientists to correct a mistake in the gene.
However, this system is often inefficient.
"The cell's usual response is not to grab a new piece of DNA, but instead to desperately try to get the ends of the two DNA strands back together," Liu said. "And in that case it can randomly delete or insert bases at the site of the break."
To overcome this hurdle, Liu's team experimented with eliminating the Cas9 enzyme's ability to break a strand of DNA. They also added a second enzyme to the system that is known to convert cytosine to uracil (C to a U). Because uracil pairs with DNA bases like a thymine (T), the change is essentially a C to a T.
"The novelty of the work is that they've fused these two proteins together to come up with a precise editing system," said Kris Saha, an assistant professor of biomedical engineering at the University of Wisconsin-Madison who was not involved with the work.
Over the course of the study, Liu and Alexis Komor, a postdoctoral researcher working in his lab, made several versions of the editing system, each improving on the last.
The first generation of their system used RNA to locate a specific sequence in DNA. It also used an inactive version of the Cas9 enzyme to bond to a strand of DNA and separate it from its partner. The second enzyme -- APOBEC1, which is derived from rats -- then went to work changing any cytosines in a small, five-base window, to uracils.
In a test tube, this system had an impressive 40% success rate. However, when the researchers tried it out in living cells, the efficiency fell to approximately 2%.
Liu said he wasn't surprised.
"When we first sketched this idea out on paper, I told Alexis we are going to have a real fight on our hands," he said. "I knew the cell was going to fight this change as hard as it could."
Over time, our cells have evolved several lines of defense to keep errors from working their way into our genome, Liu explained. Some of the hardest work that the team faced was finding ways to trick the cell into accepting the C-to-U substitutions introduced by the base editing system.
"Like any science project worthy of research, this was not easy," Liu said. "I give endless credit to Alexis Komor. Thanks to her insights and hard work, we were eventually successful."
By the time the team had created the third generation of the system, they could correct point mutations in as much as 75% of mammalian cells, Liu said.
But, of course, there is lots more work to do.
For now, the base editing system is only capable of making two types of changes in DNA -- either a C to U (essentially a T) or, with a little tweaking, a guanine (G) to an adenine (A).
The group started with the C-to-T change because APOBEC1's ability to make that change is well known, but Liu said they are currently working on adding new flavors to their base editing machine.
He also cautioned that the base editing technique is still many years away from being used to help people who have harmful genetic mutations.
"This is a first step, and an important step," he said.
He added that his group has already launched collaborations with other labs around this work.
"The genome editing field is one of the most active and vibrant, and it is our hope that researchers make use of, and improve on, base editing," he said.
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