Breakthrough gene-editing tool can find and replace DNA better than CRISPR
"Prime editing" is more precise and more efficient than CRISPR and could herald a new era of genetic manipulation.
A new tool is coming to gene editing.
The ability to edit the genes of human beings by changing the code of life -- DNA -- has dramatically improved over the last decade. Current tools, like CRISPR-Cas9 and base editors, are extremely powerful but have traditionally suffered from a lack of precision, high error rate and limited scope, hampering their ability to treat human genetic disease. A breakthrough gene-editing tool, developed by a team of researchers at Harvard University and unveiled Monday, has the ability to make extremely precise DNA edits, ushering in a new era of genetic manipulation.
"In many respects this first report is the beginning rather than the end of a longstanding aspiration ... to be able to make any DNA change in any position of a living cell or organism including, potentially, human patients with genetic diseases," says David Liu, a chemist at Harvard's Broad Institute and co-author of the new study.
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Gene editing involves changing the four bases of the DNA code, designated by the letters A, C, T and G, by deleting them, inserting them, modifying them or a combination of the three.
The research, published in the journal Nature on Oct. 21 and led by Andrew Anzalone, describes the new technique, dubbed "prime editing," in a series of elegant experiments using four human cell lines and mouse brain cells. Performing 175 different DNA edits, the researchers show prime editing can change DNA with incredible precision and, importantly, introduces errors at a much lower rate than previous gene-editing technologies. To demonstrate this, the team corrected the genetic mutations for two human genetic diseases, sickle cell and Tay-Sachs disease, in human cells. They showed the necessary edits could be made to the DNA to reverse the genetic mutations causing those diseases.
And it doesn't end with sickle cell and Tay-Sachs. Because prime editing provides one of the most precise ways to manipulate the code of life, the authors hypothesize it could enable treatment for approximately 89% of the 75,112 human genetic mutations that cause disease.
"This is a very smart piece of technology that makes gene-editing more precise," says Peter Dearden, a geneticist at the University of Otago.
To understand exactly how the complex new tool functions, it's important to understand the gene-editing tools scientists are already using -- and where the significant improvements have been made.
Old vs. new
There are two major gene-editing tools in use: CRISPR-Cas9 (or simply CRISPR) and base editors.
The CRISPR gene editing tool, often referred to as "molecular scissors," has been floated as a revolutionary tool to help eradicate species, change agricultural practices and even bring the mammoth back from the dead. Controversially, Chinese scientist He Jiankui used the technology to create gene-edited twins in November 2018, a move widely condemned by the scientific community.
CRISPR is powerful, no doubt, but it has its drawbacks. As transformative as it has been, the molecular scissors of CRISPR are rather crude. Scientists program CRISPR to seek out double-stranded DNA and make a cut across both strands. This allows for DNA to be deleted or new DNA letters to be "pasted" into the gap, but the process is significantly error-prone -- CRISPR sometimes make cuts at different points far from the target site and can introduce errors into the genes.
In November 2018, He Jiankui (pictured) claimed to have created the world's first gene-edited twins using CRISPR
On the other hand, base editors are more precise. They're sometimes referred to as "molecular pencils," because they can erase one of the four DNA letters (A, C, T, G) and write in a new letter to correct a DNA mutation. Importantly, base editors don't cut the double-stranded DNA like CRISPR does. That improves precision but comes at a cost -- they don't have the same versatility as CRISPR and only work in specific circumstances. Two papers, published in Science earlier this year, also demonstrated base editors are prone to making off-target edits.
Prime editors, continuing down the office stationery analogy, aren't scissors or pencils. Liu suggests they're much more advanced.
"You can think of prime editors to be like word processors, capable of searching for target DNA sequences and precisely replacing them with edited DNA sequences," he says.
To produce the new tool, the research team took one of the core components of the CRISPR system and fused it with a reverse transcriptase, an enzyme that can "write" the DNA code letter by letter. They join this with an engineered guide RNA, which tells the editor what letters it needs to code.
The robust tool is able to search for a DNA sequence and, sticking with the word processor analogy, either type in new letters, replace old letters with new ones or even just delete the letters altogether.
"It is versatile, and all possible types of editing (nucleotide changes, insertions or deletions) can be performed with high fidelity and high efficiency according to the paper," says Gaétan Burgio, a CRISPR geneticist at Australian National University who is not affiliated with the study.
Perhaps most remarkable is the efficiency with which the prime editor operates. One of the key improvements is the significant lack of DNA errors introduced across the range of experiments conducted by the team. This, the team hypothesizes, is because of the way prime editors interact with DNA.
Prime editors have to perform a three-step, elaborate handshake with DNA before the system allows a change to be made. CRISPR only requires a one-step handshake. In the context of the genome, CRISPR is shaking hands with basically any DNA sequence it's programmed to target. But prime editors are different. Anzalone hypothesizes the extra two steps help improve the precision of prime editors because if the handshake doesn't match up, the process is terminated.
Burgio notes that current techniques are still "quite efficient," so that gain in efficiency isn't a huge deal for current clinical research. However, he does suggest it could be quite beneficial in treating a disease like sickle cell anemia, in which the patient's cells are removed from the body, edited in a lab and then placed back into the patient with a gene edit.
And for Liu, prime editors aren't designed to take over from CRISPR-Cas9 or base editors in any way -- and in fact, they should work together.
"Each have complementary strengths and weaknesses," says Liu. "We anticipate all three classes of editing agents in mammalian cells have, or will have, roles in basic research and in applications such as human therapeutics and agriculture."
Sickle cell disease causes blood cells to take on a sticky, sickle shape, compromising their ability to transport oxygen and blocking blood flow.
Primed and ready
One of the key limitations, when the technique is ready for clinical use, will be how to deliver the editor into human cells.
"The major limitation I can see in this method is the fact that the [editor] is huge," says Burgio. "This could be problematic to deliver into the cells of living organisms for in-vivo editing."
The study in Nature showed insertion of relatively small molecules. When inserting new DNA bases, the team were able to get up to 44 DNA letters, and when deleting bases they demonstrated high efficiency up to 80 letters long. Liu suspects bigger complexes may not show the same level of efficiency and concedes that getting the genetic edits into humans is a challenging prospect.
"Delivery always remains an important consideration and a challenge, so our lab, and others, are working on this problem very hard and we hope to be able to deliver prime editors into animals in the near future," says Liu.
With the release of the paper, researchers from around the world will now have the opportunity to test prime editing in a range of different cell types and models. A clearer understanding of the tool's limitations and benefits will become more apparent in the future, just as they did with CRISPR and base editors before them. Anzalone, who pioneered much of the research, is really excited about it.
"We've been working on this for over a year, so it feels great to finally be able to share it with the scientific community," he says.
