Single genome-editing strategy promises to treat multiple disorders

Genetic disorders often stem from small errors in the DNA sequence with major consequences. Many diseases like cystic fibrosis and Batten disease can be traced to changes disrupting the cell’s ability to build a complete, functional protein. One particularly common culprit is the nonsense mutation, where a single incorrect DNA letter inserts a premature stop signal. When the cell encounters it, protein production ends too early, leaving the body without important enzymes, transporters or structural components.

Nonsense mutations account for about a quarter of all known disease-causing genetic changes. Each one halts a different protein at a different point, creating a wide range of disorders that, at present, require separate treatments. Each therapy needs to be designed, tested and approved on its own. This is a slow and expensive process.

A study in Nature recently revealed a way around this challenge. Instead of crafting a therapy for every mutation, researchers from the Broad Institute, Harvard University, and the University of Minnesota have developed a method to address many nonsense mutation diseases using a single genome-editing strategy. Their approach, called Prime-Editing-mediated Readthrough of premature Termination codons (PERT), reprogrammes one of the cell’s own genes into a tool to override premature stop signals, allowing the cell to ignore the faulty instruction and complete the protein.

“This study offers an intriguing proof-of-concept for a gene-agnostic therapy that could, in principle, benefit many rare diseases caused by nonsense mutations,” Debojyoti Chakraborty, senior principal scientist at CSIR-Institute of Genomics and Integrative Biology, New Delhi, and who wasn’t involved in the study, said.

Repurposing genes

Cells make proteins by transcribing the DNA into mRNA, written in a sequence of three nucleotides at a time; each set of three is called a codon. Then tRNA acts like a translator: each one recognises a specific codon and transports the matching amino acid, like making a photograph from its negative. Finally, a cellular machine called the ribosome strings these amino acids together, one by one, to make proteins.

The tRNA genes number in the hundreds. Many of them are redundant because they perform overlapping functions, so the loss or alteration of one of them is often harmless.

The researchers used this redundancy to test whether a non-essential tRNA gene could be edited into a suppressor tRNA — a molecule that reads through premature stop signals and inserts an amino acid there instead. Laboratories have used natural suppressor tRNAs for decades but they’ve been unsuitable for therapies thus far due to concerns about their safety and durability.

Using a precise genome-editing approach called prime editing, the team showed that a human tRNA gene can be rewritten to permanently operate as a suppressor tRNA while also producing tRNA at safe, natural levels. This allowed the edited cell to override premature stop codons and make full-length proteins without disrupting global protein production.

Finding effective candidates

Human cells contain 418 tRNA genes. With the help of prime editing, the researchers found that four tRNAs — called leucine, arginine, tyrosine, and serine — showed promise to suppress a premature stop codon called TAG. However, the natural versions of these tRNAs weren’t good enough for therapeutic use.

To increase their effectiveness, the researchers engineered thousands of variants of the four tRNA by adjusting their DNA sequences and by making small changes to the tRNA structure itself. These improvements made the tRNAs more stable and better at decoding premature stop signals. This multi-step engineering effort produced several optimised suppressor tRNAs.

The next challenge was to install them efficiently into the genome. However, editing a tRNA gene is difficult because that part of the DNA is often compact and tightly folded, making it harder for genome-editing enzymes to access it.

To overcome this, the researchers turned to the specifics of prime editing. This technique uses a specialised molecule called a prime-editing guide RNA, or pegRNA, to lead the editing machinery to the correct spot on the DNA and hold the template needed to write the new genetic code.

Because the success of this process depends heavily on the precise design of the pegRNA, the team created a library of more than 17,000 different ones and tested various configurations to identify the ones that could successfully access the tightly folded DNA and rewrite the native tRNA gene into its optimised suppressor form.

Based on the results of this screen, the team identified a prime-editing enzyme that they named PE6c. It proved especially effective at rewriting the targeted DNA sequence, and became more efficient when paired with a strategy called PE3 — which uses an additional guide RNA to steer the cell’s repair machinery to adopt the edited sequence.

In cultured human cells, this combination had 60-80% editing efficiency, which is unusually high for multi-base genomic edits. To compare the standard method for precise gene insertion, called homology-directed repair, is typically 10-20% efficient, or below, in similar contexts.

Safety tests indicated the process didn’t accidentally alter unrelated parts of the DNA, didn’t disturb the cell’s overall activity or normal protein production, and it distinguished between faulty and correct instructions. In particular, it ignored the premature stop signals causing the disease while still respecting the natural stop signals that mark the actual end of a protein.

The researchers called this complete package PERT. To evaluate its therapeutic potential, they tested the method in cell models of Batten disease, Tay-Sachs disease, and Niemann-Pick C1 disease, all caused by premature stop codons.

After installing the engineered suppressor tRNA, enzyme activity in the Batten and Tay-Sachs models rose to 17-70% of their normal levels. In Niemann-Pick C1 models, cells produced measurable amounts of full-length NPC1 protein, which is otherwise absent when there’s a nonsense mutation.

Results in mice

To evaluate PERT in a living organism, the team used AAV9 to deliver the prime-editing components into newborn mice. AAV9 is a common gene-therapy vector, a harmless virus repurposed as a microscopic delivery vehicle to ferry genetic cargo into cells. The goal was to use it to convert a natural mouse tRNA gene into a suppressor tRNA in vivo and assess its ability to restore protein production.

In the Hurler syndrome mouse model, PERT restored 1.7-7% of normal enzyme activity in the brain, heart, and liver. While modest, these levels are known to meaningfully reduce disease severity. Treated mice also showed better cellular pathology and no signs of toxicity.

“The authors present strong laboratory evidence showing that their engineered tRNA approach can restore protein function in multiple models, which is an important advance,” Dr. Chakraborty said.

But he also emphasised the practical limitations: “Key challenges remain, particularly around delivery, long-term safety, and performance across different tissues, before this strategy can realistically move toward patients.”

Yet these early successes have offered some momentum. The first clinical use of base editing in an individual reported earlier this year involved a TAG stop codon. The case showed that established delivery methods like viral vectors can carry gene-editing tools into the necessary tissues. This means PERT has a viable path to the clinic.

Manjeera Gowravaram has a PhD in RNA biochemistry and works as a freelance science writer.