A New Gene Editing Tool Could Make CRISPR More Precise

By now, you’ve probably heard of CRISPR technology, the “molecular scissors” that snip DNA at a target sequence and enable genetic editing. CRISPR revolutionized medical science by introducing an easy-to-use way to modify the genome, but it’s not the only tool at scientists’ disposal. In a paper published in Nature today, researchers added a new technique to the genetic editing toolbox: prime editing.

If the original CRISPR mechanism is like a pair of miniscule scissors cutting up a sentence of the DNA code, “you can think of prime editors to be like word processors, capable of searching for precise DNA sequences and replacing them,” says David Liu, the chemical biologist at the Broad Institute and Harvard University who led the research. Where the familiar CRISPR technique fully cleaves a strand of DNA in two, often creating some tiny, inadvertent genetic changes as byproducts, prime editing begins by slicing just one of the two strands of the double helix. The method is sleeker, less invasive, and offers the potential for precision genetic editing.

Traditional CRISPR is hardly low-tech; it’s a Nobel Prize-buzzworthy process so minute that it’s completely invisible without a high-powered microscope. Still, as Megan Molteni wrote for Wired last year, “CRISPR Classic is somewhat clunky, unreliable, and a bit dangerous. … If the Model T was prone to overheating, CRISPR Classic is prone to overeating.”

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CRISPR-based editing makes use of a cellular defense mechanism that originated in bacteria to scan for viral DNA and then dice it up. Once the system recognizes the sequence of bases (the “letters” that make up the DNA alphabet) it’s been instructed to look for, it can cleanly cut the two-part DNA strand, creating what’s known as a double-strand break. The cell detects and swoops in to repair this damage with whatever genetic materials it has available, often a snippet of donor DNA the scientists have inserted into the cell alongside CRISPR. However, the repair process might also rope in some stray letters or chop off pre-existing slivers of the genome. These insertions and deletions, called “indels,” are a frequent consequence of the classic CRISPR process.

Indels aren’t always a problem. If you simply snip DNA in the middle of a target gene and let it repair itself, the resulting indels will often deactivate that gene. But they’re unpredictable. Because DNA is processed in sequences of three letters at a time, indels can also offset the genetic code for a particular protein, changing the output of a cell. And if too many double-stranded breaks were to be induced at once, they could prove toxic to the cell. If the “goal is to minimize perturbing the cell or patient beyond making the desired edit,” Liu explains in an email, “creating a mixture of products such as indels is typically undesirable.”

CRISPR editing to insert a particular genetic sequence, meanwhile, can leave donor DNA floating in the cell. At the moment, the downstream effects of those fragments are still unclear, says Le Cong, an assistant professor at Stanford who worked on some of the Broad Institute’s early CRISPR research.

Prime editing is the most recent of the tools developed to address these limitations and fine-tune the genetic editing process. It employs the same mechanism as traditional CRISPR to pinpoint the location of a given genetic sequence and guide molecular tools there. For this reason, Cong considers the new tool of prime editing, which he calls “groundbreaking,” a new category of CRISPR-based editing.

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The cut-and-repair mechanism is where prime editing really differs. Every prime editor (PE) contains multiple enzymes fused into one long, multipurpose piece of RNA. After the prime editor hones in on the genetic target, it makes a cut in one strand—not two—of DNA. Then, yet another part of the PE molecule finds the just-snipped end of the DNA and extends it, manufacturing an edited DNA sequence from a template. The new DNA instructions are created by a reverse transcriptase, a class of enzyme most familiar as the mechanism through which retroviruses like H.I.V. integrate themselves into a host cell’s genome.

With the new, customized sequence of DNA manufactured, the cell repairs itself, trimming off the old fragment of DNA and sealing in the new one. When the cell realizes that there’s a mismatch between the edited sequence and the strand opposite it, it will edit the previously unaltered strand so both halves of the helix adopt the change. “It is a very elegant technique that has pretty broad applications,” Cong says.

Liu and Andrew Anzalone, a researcher who also oversaw the project, designed several different iterations of the prime editing system. To encourage the cell to mirror the edit in both strands, systems called PE3 and PE3b also nick the unedited strand of DNA to kickstart the cell’s repair mechanisms.

The scientists tested different versions of the prime editing approach in four human cell types as well as mouse neurons. Efficiency rates varied, but Liu says that for the most part, prime editing proved as efficient, if not more, at making small edits than the more traditional approach of creating and then patching double-strand break. It also produced far fewer indels. Prime editing with the PE3 systems made the correct edits up to 50 percent of the time, a rate Cong considers “very efficient” for genetic editing.

Prime editing isn’t the first or only tool scientists have to edit DNA without creating double-stranded breaks. In 2016, Liu’s lab debuted base editing, which chemically swaps one base, or DNA letter, for another. Under certain conditions, base editing proved more efficient than prime editing, but it can’t be used in as many situations. Base editing can only make four types of point edits, and only a small window of genetic material is accessible once the editor binds with the DNA, says Alexis Komor, who worked on base editing alongside Liu and now leads her own chemical biology lab at the University of California, San Diego.

Each genetic modification tool is best suited to make different changes. Liu’s team used prime editors to cut out the four extra bases that cause Tay-Sachs disease and fix the single base that causes sickle cell disease, both genomic alterations that base editing can’t accomplish and traditional CRISPR editing can’t do without potentially damaging double-strand breaks. But edits where larger sections of genetic material need to be removed or added, like forms of hereditary heart disease, are outside of prime editing’s range, so double-stranded breaks remain the right approach, Cong says.

With any new genetic editing technology comes concern over inadvertent changes. Liu, Anzalone and their team tested 16 sites on the genome where the Cas9 enzyme is particularly prone to accidental edits, and they found that prime editing only revised three, making its off-target change rate only a fraction of classic CRISPR’s. This smaller error rate may be due to the fact that prime editing requires three pairing events—three lock-and-key matches—to complete its work, not just one. Still, Liu acknowledges that a whole-genome analysis of prime editing will be necessary in the future, something his lab is working on.

The paper in Nature represents a first step for future research. Liu says that it’s “really important that the community test and, if needed, optimize prime editing in as many types of organisms as possible.” (The technology will be accessible through the non-profit DNA library Addgene.)

Another question for researchers: What’s the best way to get a prime editor, a super-sized “macromolecule,” as Liu put it, into cells in living organisms, not test tubes? Short-term, Komor says, prime editing, like base editing, will help labs like hers study small mutations that might cause disease. Looking further into the future, once prime editing has been given many more trial runs, the technology could offer therapeutic treatments for medical conditions, too. By Liu and Anzalone’s estimate, at least 89 percent of known disease-linked genetic mutations could theoretically be corrected using prime editing.

Komor calls prime editing “a really cool addition to the genome editing toolbox.” But just as this new technique offers a refinement of the approach pioneered in 2012, prime editing is also a jumping-off point for future innovation. “Everyone,” Komor says, “needs to start working on this: How do we [simultaneously] modify both strands?”

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