Prime-Editing-mediated Readthrough of premature Termination codons (PERT)

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.

Prime-Editing-mediated Readthrough of premature Termination codons (PERT) is an innovative gene-editing strategy designed to treat genetic diseases caused by Nonsense Mutations.

A nonsense mutation creates a Premature Termination Codon (PTC), which acts like an early “stop” sign in the genetic code. This results in truncated, non-functional proteins, leading to disorders like Cystic Fibrosis, Duchenne Muscular Dystrophy, and certain cancers.

How PERT Works?

PERT utilizes Prime Editing (PE)—a “search-and-replace” genome editing technology—to bypass these stop signs without necessarily “fixing” the original mutation to its wild-type state.

 The Mechanism:

Instead of traditional CRISPR which cuts DNA, PERT uses a nicking Cas9 fused to a Reverse Transcriptase (RT).

• Targeting: A pegRNA (prime editing guide RNA) directs the enzyme to the PTC site.

• The “Readthrough” Edit: Instead of reverting the stop codon (e.g., TAG) back to the original amino acid, PERT introduces a specific synonymous or “near-cognate” codon that the cellular machinery recognizes as an instruction to keep building the protein.

• Flanking Sequence Optimization: PERT often involves subtly altering the sequences around the stop codon to decrease the “termination efficiency,” making it easier for the ribosome to skip over the error.

Key Advantages over Traditional Methods:

• Precision: Unlike “Readthrough Drugs” (like Gentamicin), which cause the ribosome to skip stop codons randomly throughout the body, PERT is site-specific. It only affects the mutated gene.

• No Double-Strand Breaks: Traditional CRISPR-Cas9 creates breaks in DNA that can lead to unintended deletions or insertions (indels). PERT nicks only one strand, making it significantly safer.

• Permanent Fix: While mRNA therapies or drugs require lifelong administration, a single PERT intervention could theoretically provide a permanent cure by modifying the genomic DNA.

Challenges and Issues:

• Efficiency: Prime editing is currently less efficient than base editing or standard CRISPR in certain cell types. Getting enough “readthrough” to restore therapeutic levels of protein is a hurdle.

• Delivery: Like all advanced gene therapies, delivering the large Prime Editing machinery into the specific target organs (like the lungs for Cystic Fibrosis) remains a primary challenge.

• Codon Choice: Choosing the wrong replacement codon might result in a protein that is full-length but “misfolded” or less active than the original.

Way Forward:

The development of PERT marks a shift from “correcting” mutations to “functional bypassing.” Future research is focusing on:

1. Engineered pegRNAs: To increase the success rate of the reverse transcription.

2. Viral and Non-viral Vectors: Using Lipid Nanoparticles (LNPs) or AAV vectors to improve delivery to human tissues.

3. Combination Therapy: Using PERT in conjunction with existing small-molecule readthrough enhancers to maximize protein production.

 Conclusion:

PERT represents a sophisticated middle ground in genomic medicine. By focusing on protein restoration rather than perfect sequence correction, it simplifies the “search-and-replace” requirements of gene editing, offering a potent new weapon against the thousands of genetic diseases caused by premature stop codons.