CRISPR has undoubtedly revolutionized genetic engineering. However, the original version could introduce unwanted and potentially hazardous changes to the genome by causing double-stranded breaks to DNA. Later iterations of the tool, base and prime editing, could overcome this limitation. Biochemist David Liu from Harvard University recently won the 2025 Breakthrough Prize for developing these technologies. While base editing is useful for point mutations, prime editing offers a wider range of applications, such as multi-nucleotide insertions and deletions. Discover what prime editing has to offer in this article.
Next-Generation Prime Editors May Fix Pathogenic Repeats
Like base editing, prime editing offers a safer way to genome editing by relying on a nickase enzyme that “nicks” one DNA strand at a time, rather than cutting both simultaneously. Then, with the addition of a reverse transcriptase and a special guide RNA, called prime editing guide RNA (pegRNA), this tool can perform edits beyond single-base changes—think CRISPR 3.0. However, first-generation prime editors couldn’t fix sequences longer than ten base pairs. So, Liu and his colleague, Andrew Anzalone, developed yet another iteration of the technology, called twin prime editing, which uses two pegRNAs instead of one and can edit sequences up to 100 base pairs long. The researchers hope that next generations of the tool could edit even longer sequences and tackle diseases that arise due to pathogenic sequence repeats, like Huntington’s disease.
THE STEPS OF PRIME EDITING | |||
 | The prime editing machinery comprises a prime editing guide RNA (pegRNA) and a Cas9 nickase enzyme fused to a reverse transcriptase. |  | |
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 | The pegRNA binds to a matching sequence on the target DNA strand. |  | The Cas9 nickase cuts the unbound complementary strand, creating a flap. |
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 | The dangling DNA flap binds to a matching sequence on the other end of the pegRNA. |  | The reverse transcriptase extends the flap by filling in the desired insertion sequence from the template next to the binding site. |
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 | The edited flap competes with and displaces the flanking nucleotide sequence (right), which is ultimately removed by the cell. |  | The opposite strand is nicked and repaired to match the newly edited flap, completing the edit. |
Base and Prime Editors Still Have Toxicities
Base and prime editing are safer than the original version of CRISPR because they don’t induce error-prone double-stranded DNA breaks. However, no technology is perfect. Recently, gene therapy researcher Luigi Naldini and his colleagues showed that base and prime editing still occasionally introduced double-strand breaks and with them, unwanted mutations, though these occurred at much lower rates than their earlier counterpart. As more CRISPR therapies enter the clinic, scientists encourage a more thorough examination of their risks.
Virus-Like Particle-Delivered Prime Editors Fixed Mice Vision Loss
Prime editing offers unparalleled precision, but this comes at the cost of a more challenging delivery into cells. Engineered virus-like particles (eVLP) can help researchers overcome this technical limitation. Recently, Liu’s team used eVLP-delivered prime editors to correct vision loss in a mouse model of genetically inherited retinal degeneration. In a previous iteration of this system, the delivered prime editors had poor editing efficiency, which the new tool managed to improve by about 65-fold.
A new tool tracks cells’ gene expression history by using barcoded cis-regulatory elements to deliver prime editing guide RNAs to specific genomic locations.
©iStock, bymuratdeniz
A Prime Editing-Based Tool Tracks Gene Regulation Dynamics Over Time
Gene expression dynamics offer a glimpse into the lives of cells. However, existing technologies could only study gene regulation at any single point in time. Recently, Jay Shendure, a geneticist at the University of Washington, and his colleagues developed a method to record gene regulation history by cis-regulatory elements (CREs), stretches of DNA sequence that regulate gene transcription. Before a gene is turned on, transcription factors bind to CRE to start transcription. Shendure’s tool relies on individually barcoded CREs that deliver pegRNAs to specific locations in the genome, allowing researchers to monitor how multiple CREs mediate gene regulation over time.
Prime editing offers hope for children with alternative hemiplegia of childhood (AHC), an extremely rare and severe neurodevelopmental disorder.
©iStock, Viktor Aheiev
Prime Editing Corrected Rare Neurodevelopmental Disorder in Mice
Alternative hemiplegia of childhood (AHC) is an extremely rare and severe neurodevelopmental disorder. It manifests as recurrent bouts of paralysis on one side of the body (hemiplegia), involuntary muscle contractions, and seizures. This disease currently has no treatment, leaving patients with muscle loss, developmental delays, and cognitive impairments. Recently, rare disease researcher Cathleen Lutz of the Jackson Laboratory, Liu, and their colleagues used prime editing to correct five different mutations in ATP1A3, both in patient-derived stem cells and a mouse model of the disease. ATP1A3 encodes a transmembrane ion pump, and mutations in this gene account for up to 70 percent of AHC cases. While more research is needed, these findings extend hope for patients of this incurable disease and their families.
