Background

Existing third-generation CRISPR-Cas9 gene scissors functioned by completely cleaving the target DNA double strand. The intracellular non-homologous end joining (NHEJ) repair pathway induced in this process often resulted in unintended random base deletions or insertions. Particularly when introducing therapeutic genes of several kilobases (kb) or attempting to correct structural variations like chromosomal inversions or translocations, the risk of chromosomal breakage or large-scale deletions due to ligation errors at the cleavage site remained a constant threat.

To reduce these side effects, Prime Editing (PE) technology, which incorporates reverse transcriptase, emerged; however, early platforms were limited to small edits within a few dozen base pairs. Despite the advantage of recording foreign sequences at target positions without double-strand breaks, there were clear physical limitations for large gene insertions or chromosomal remodeling. Complex platforms incorporating recombinases also failed to accelerate their clinical advancement due to hurdles such as reduced binding site recognition efficiency and exceeding carrier loading capacities.

Key Findings

The Prime Assembly platform reported here directly addresses the limitations associated with genomic structural variations. Researchers employed a strategy of precisely combining engineered prime editing complexes with multiple prime editing guide RNAs (pegRNAs) to simultaneously target multiple specific sites. This enables chromosomal engineering—such as inverting (inversion), duplicating (duplication), or repositioning (translocation) target sequences of tens of kilobases without external donor DNA or double-strand breaks.

Experimental data shows that in human cell lines and primary immune cell models, the efficiency of correcting DNA inversions larger than 20 kb increased approximately 3.8-fold compared to conventional Twin Prime Editing (TwinPE). During the induction of chromosomal inter-chromosomal translocations, the measured off-target cleavage and chromosomal fragmentation rates decreased by more than 95% compared to the conventional Cas9-treated group. Because multiple pegRNAs were designed to work complementarily, unintended fragment deletions resulting from single-target escape events were suppressed to less than 1%.

Significance and Outlook

Prime Assembly opens the possibility for fundamental treatment of intractable structural variation genetic diseases, such as the 140kb inversion that is a major cause of Hemophilia A or large exon deletions in Duchenne Muscular Dystrophy (DMD). As cleavage toxicity is minimized, the safety of manufacturing processes for cell and gene therapies based on hematopoietic stem cells or induced pluripotent stem cells (iPSCs) is also expected to be significantly strengthened. In the field of synthetic biology, precise genome design is anticipated to enable the simultaneous rearrangement of large-scale metabolic pathway gene clusters.

However, the challenge of optimizing in vivo delivery vehicles remains for clinical application. A system must be established to safely deliver multiple accessory factors and guide RNAs by partitioning them into single non-viral lipid nanoparticles (LNPs) or adeno-associated viruses (AAVs), and follow-up studies on off-target mutations related to long-term storage and in vivo expression persistence must continue.