Engineered Prime Editors with Improved Precision Could Provide Safer Cell and Gene Therapies

Prime editing is an attractive alternative to CRISPR-Cas9 gene editing because it can rewrite sections of DNA without the risks associated with introducing double-stranded breaks.¹ However, prime editing can still introduce errors, such as insertion and deletion mutations (indels), presenting a key obstacle to its adoption in cell and gene therapies.¹

In a new study published in Nature, researchers from the Massachusetts Institute of Technology (MIT) showcased engineered prime editors with significantly lower rates of indel formation. “This paper outlines a new approach to doing gene editing that doesn’t complicate the delivery system and doesn’t add additional steps, but results in a much more precise edit with fewer unwanted mutations,” geneticist and coauthor Phillip Sharp remarked in a press release.

Prime editing uses a Cas9 nickase (Cas9n) fused to a reverse transcriptase (RT) enzyme, along with a prime editing guide RNA (pegRNA) that contains both the target sequence for editing and the replacement sequence. Once Cas9n locates the genomic target, it creates a single-stranded break or ‘nick’ in the 3’ strand of DNA, which can then anneal to the pegRNA. The RT enzyme then goes to work writing a new extension to the 3’ strand based on this template.

This final step is where potential errors can occur; the new, edited 3’ strand of DNA is now mismatched with the complementary strand, meaning that the 5’ can outcompete and displace it, limiting editing efficiency. If this occurs, the edited 3’ strand can be integrated into unintended positions, resulting in large deletions and other unwanted alterations to the genome.

To address these limitations of prime editing (PE) systems, the authors combined several different strategies. Other researchers had previously shown that Cas9n releases the 3’ end of nicked DNA while remaining bound to the 5’ end, so the MIT team first wanted to destabilize the ends of nicked DNA, allowing them to be degraded so the new strand could be integrated into the genome.²

Fortunately, the authors had discovered in an earlier study that mutations in Cas9 can modify where nicks occur.³ By creating mutated Cas9n variants and testing them in HEK293T cells, the team observed that these mutants resulted in relaxed nick positioning and degradation at the ends of nicked DNA. They also found that some of the mutated PEs had much lower indel error rates than the commonly used PEmax system. By combining some of the mutations, they created a precision prime editor (pPE) system with a 118-fold lower indel error rate and an enhanced edit:indel ratio.

While this pPE system had a much lower indel error rate, it had a slight reduction in editing efficiency, prompting the team to introduce mutations that are known to increase the activity of Cas9n. After initial testing in HEK293T cells, the authors again found that a combination of these mutations resulted in maximal editing efficiency. The best-performing variant, which they named extra-precise prime editor (xPE), had an edit:indel ratio of 354:1, a significant improvement compared to pPE’s ratio of 276:1.

Because the xPE system still had slightly lower editing efficiency compared to PEmax, the final step was to boost its performance. The team hypothesized that protecting the unstable 3’ ends of the pegRNA molecule from degradation would increase editing efficiency in their xPE system, particularly since it has reduced overlap between the pegRNA and the nicked 3’ DNA ends. Using a La poly-U RNA-binding protein, the authors created the very-precise prime editor (vPE) system, which had a 3.2-fold boost in editing efficiency compared to xPE, and an increased indel:error rate of 465:1.

The authors suggested in the discussion of the paper that their vPE system can be easily integrated into existing genome editing applications, providing an attractive alternative for the development of cell and gene therapies. “For any drug, what you want is something that is effective, but with as few side effects as possible,” said coauthor and biotechnologist Robert Langer in the press release. “For any disease where you might do genome editing, I would think this would ultimately be a safer, better way of doing it.”