Background

In the field of genome editing, the challenge has long been to precisely insert a wide range of DNA sequences into the genome without inducing double-strand breaks (DSBs). Conventional CRISPR-Cas9 technology relies on cutting both DNA strands and cellular repair mechanisms, which can lead to unintended mutations. To overcome this, Prime Editing technology was developed, which avoids DSBs and is safer, but it has a limitation in that it can only insert DNA sequences up to a few dozen base pairs at a time.

To insert large DNA fragments into the genome, researchers have combined recombinases or transposases. However, these methods require multi-step editing, are inefficient, and leave behind unwanted sequence footprints, making clinical application difficult. A new approach was urgently needed to insert large genetic information safely and precisely in a single step.

Key Discovery

To overcome these limitations, the research team developed a new method called Donor-Complementary Prime Editing (DoPE). This system combines a 3'-overhang double-stranded DNA (odsDNA) donor carrying the target genetic information, a pair of overhang-complementary prime editing guide RNAs (opegRNAs) that bind to the donor's overhang sequences, and a PE2* prime editor (PE2*).

DoPE improves efficiency by using odsDNA donors with short overhangs of approximately 30 nucleotides. The research team experimentally demonstrated that it can precisely insert genetic material ranging from small fragments to as large as 12.5 kilobases (kb). This means that large genetic information can be inserted into the genome in a single reaction without the need for additional recombinases.

To validate the performance of DoPE, the research team conducted in situ saturation mutagenesis experiments targeting a region of the Enhanced Green Fluorescent Protein (EGFP) gene. Using a pair of opegRNAs and a donor pool constructed from synthesized single-stranded oligonucleotides, they successfully created a precise mutation library at both amino acid and nucleotide resolutions.

Furthermore, the team tested the correction of PRKCSH gene mutations using DoPE technology. In experiments replacing damaged exon regions of the gene, the researchers simultaneously or individually replaced various mutant alleles with normal sequences. This achievement is considered a foundation for mutation-agnostic therapy, which can insert normal genes regardless of the type of mutation.

Significance and Prospects

DoPE technology overcomes both the large DNA insertion limitations of existing prime editing and the risks associated with genome cutting. It eliminates the need to design custom guide RNAs for each individual mutation, making it possible to develop a universal gene therapy that inserts intact large gene blocks. By excluding external factors such as recombinases, it also offers significant advantages in terms of in vivo safety for clinical applications.

However, there are still challenges to be addressed before this technology can be applied in clinical settings. It must be verified whether the high editing efficiency demonstrated in cell experiments is consistently achieved in living animals. Integration with in vivo delivery technologies capable of safely delivering large genetic material and protein complexes to disease target sites is also a challenge. Long-term studies are also needed to precisely track whether unintended off-target effects occur during the insertion of large genetic material.