Early results suggest that prime editors are cleaner than CRISPR–Cas9 and more versatile than base editors, but many questions remain.

A paper recently published in Nature from David Liu and co-workers discloses a ‘prime’ gene-editing system many years in the making.

In principle, the system, comprising a catalytically impaired Cas9 enzyme and an engineered reverse transcriptase, may be able to address ~89% of the human genetic variants known to be pathogenic. Already, it has been snapped up as the key founding intellectual property for Cambridge, Massachusetts-based startup Prime Medicine. Another startup, Beam Therapeutics, has also moved to gain exclusive rights to use of the technology in editing single-base transition mutations, as well as for developing therapies for sickle-cell disease. In return, Prime Medicine has access to aspects of Beam’s CRISPR base-editing technology, including its Cas12b enzyme for use in mammalian systems, and to its delivery technology. Prime editing is still a nascent, albeit highly promising, technology. Demonstrating its feasibility in a wide range of cells and tissues will be key to its future development.

The prime-editor protein is a fusion protein of Cas9 and reverse transcriptase, which together form a complex with a ‘prime-editing guide RNA’ (pegRNA). The pegRNA directs the complex to the target site and also acts as a carrier of the desired edit. The Cas9 ‘nickase’ component of the prime editor can be programmed to bind a target DNA sequence and introduce a single-strand cut, rather than the double-strand break that wild-type Cas9 enzymes make. The pegRNA also carries a primer binding site that allows hybridization to occur between the 3′ end of the nicked DNA strand and the pegRNA, which then acts as a template for the reverse transcriptase component of the prime editor. The reverse transcriptase then catalyzes the transfer of the genetic information encoding the edit into the host cell’s genome. A series of DNA cleavage and ligation steps catalyzed by the cell’s endogenous DNA repair machinery eliminates the redundant single-stranded DNA flaps generated during the process. “The key to prime editing’s versatility is that the part of the pegRNA that specifies the edited DNA sequence can be virtually any sequence,” says Liu by e-mail.

Liu, of the Broad Institute and Harvard University, spearheaded Beam’s base editor technology—a modified, RNA-programmable Cas9 protein fused to a base-editing deaminase enzyme that acts on single-stranded DNA. Between its adenine base editor (ABE), which converts adenine to inosine, and its cytosine base editor (CBE), which converts cytosine to thymine, Beam’s platform can accomplish all four possible single-base transition mutations. But prime editing appears to be a far more general approach, being capable of introducing to the target genome the same four single-base transition mutations and all eight possible single-base transversion mutations (purine to pyrimidine or vice versa), as well as precisely targeted insertions and deletions.

The prime system also offers advantages over traditional gene editing via conventional CRISPR–Cas9. CRISPR–Cas9 edits rely on the cell’s own repair mechanisms—non-homologous end joining (NHEJ) or homology-directed repair (HDR)—to fix the break. These DNA repair mechanisms generate an uncontrollable and undesirable mix of editing byproducts, such as random insertions or deletions (indels), as well as other genetic rearrangements, which complicate the retrieval of those cells carrying the correct edit. The prime editing system doesn’t rely on NHEJ and HDR, but it still requires other intrinsic repair pathways, such as DNA mismatch repair, to be sufficiently active in cells. Although the prime system does not require a double-stranded DNA break and initial results from the Nature paper suggest it has fewer undesirable off-target and on-target effects than CRISPR–Cas9, random indels still occur.

Prime editing introduces far fewer off-target instances when measured at known off-target Cas9 sites. This is most likely because its mechanism involves three separate DNA binding events (between the guide RNA and the target DNA, between the pegRNA primer binding site and the target DNA, and between the 3′ end of the nicked DNA strand and the pegRNA, which then acts as a template for the reverse transcriptase component of the prime editor. The reverse transcriptase then catalyzes the transfer of the genetic information encoding the edit into the host cell’s genome. A series of DNA cleavage and ligation steps catalyzed by the cell’s endogenous DNA repair machinery eliminates the redundant single-stranded DNA flaps generated during the process. “The key to prime editing’s versatility is that the part of the pegRNA that specifies the edited DNA sequence can be virtually any sequence,” says Liu by e-mail.

Overall, prime editing looks like a useful new addition to, rather than a replacement for, existing gene-editing techniques. “It looks very promising for relatively small genetic alterations,” says Alexander Marson of the University of California, San Francisco. “One question is, will this be adaptable to make larger genetic alterations?” Marson’s lab has developed, in collaboration with CRISPR pioneer Jennifer Doudna, a CRISPR–Cas9 system that enables reprogramming of primary human immune effector cells. This can involve the insertion into the target genome of several kilobases of DNA. “It’s not immediately obvious that this approach would work for these kinds of larger targeted insertions,” he says. So far, Liu says, prime editing has generated insertions of up to 44 base pairs and deletions of up to 80 base pairs, but it is not yet clear whether the technology can make larger alterations. “We haven’t tried using prime editing for very long (gene-sized) insertions or deletions, but based on its mechanism I suspect modifications to the basic prime editing scheme would be needed for such changes to be efficient,” Liu adds.

Liu himself notes that although base editing is more limited in its scope, it is more efficient and generates fewer indel byproducts. Base editors are also more restricted by the need for a protospacer-adjacent motif (PAM)—a short sequence of nucleotides required for successful Cas9 recognition of the target site—to be located within a certain range of the target base. Liu and colleagues reported that the 179 different edits they performed at twelve loci in the human and mouse genomes ranged from being three base pairs upstream to 29 base pairs downstream of a PAM. For successful base editing, the target base needs to be located within an ~5 base pair window about 13–15 base pairs from a PAM sequence.

The Nature paper described the use of prime editing in four human cancer cell lines and in postmitotic primary mouse cortical neurons. “The efficiency of prime editing varied quite a bit across these cell types, so illuminating the cell-type and cell-state determinants of prime editing outcomes is one focus of our current efforts,” Liu says. “Our understanding of the cellular factors that determine prime editing efficiency is incomplete at this point, so it’s difficult to predict which cells will be better or worse suited for prime editing.”

As with other gene-editing systems, another aspect that will be critical to understand for translation is how best to deliver the prime system to a wide range of cells and tissues. “It is an important aspect of performance that has yet to be demonstrated,” says Edward Rebar, chief technology officer at Sangamo Therapeutics, which has pioneered the use of engineered zinc finger nucleases in genome editing. Liu’s group employed either transfection or an electroporation-based delivery system to transfer the prime editing components into the four human cell lines it tested, but used a lentiviral vector to transduce the mouse cells. Delivery of prime editors for in vivo applications will be complex, as the components of a prime editor exceed the packaging limits of standard adeno-associated viruses, which are the most convenient vectors for genome editing. “It is going to be difficult,” says Rebar. The task is not impossible, he adds, given the advances made in adeno-associated virus engineering.

Given prime editing’s early stage of development, any assessments of its strengths and weaknesses are inevitably provisional. In the three-and-a-half years since the development of base editing, says Liu, the technique has been widely used, in organisms ranging from bacteria to plants, mice and primates. The Addgene not-for-profit plasmid repository has distributed DNA blueprints for base editors over 7,500 times to over 1,000 researchers around the world. “While we are very excited about prime editing, it’s brand new and there has only been one paper published thus far. So there’s much to do before we can know if prime editing will prove to be as general and robust as base editing has proven to be,” says Liu.