Bottleneck in multiplexed guide processing and the limitation of Csy4 self-toxicity in Prime Editing Prime editors (PE), which induce substitutions, insertions, and deletions without double‑strand DNA breaks, have become a core tool for plant genome engineering. However, a severe bottleneck arises when multiple prime editing guide RNAs (pegRNAs) must be liberated and processed from a single transcript for multiplexed targeting. Introducing the endonuclease Csy4 to cleave spacers between pegRNA sequences alleviated this, but Csy4’s intrinsic hyperactive RNA cleavage caused unwanted transcript degradation and pronounced cellular toxicity in the model plant Arabidopsis thaliana, crippling both transformant survival and heritability.

‘Uncleavable Fusion’ molecular architecture: securing stability by disabling cleavage The research team engineered a breakthrough by chemically modifying the catalytic residues of Csy4 so that it retains RNA‑binding and structural recognition while losing its indiscriminate cleavage activity, rendering it ‘uncleavable.’ This engineered Csy4 was physically repositioned and fused directly to the prime‑editor protein complex, creating an ‘Uncleavable fusion’ architecture. The system scaffolds the three‑dimensional structure of pegRNAs and dramatically reduces the cytotoxic side‑effects of native Csy4.

200 % increase in prime‑editing efficiency and demonstration of next‑generation heritability in Arabidopsis Application of the fusion platform in Arabidopsis resulted in more than a two‑fold (≥200 %) boost in precise editing efficiency at the intended loci compared with previous hybrid systems. Importantly, the abrogation of Csy4 toxicity preserved normal plant developmental programs, allowing corrected genotypes to be transmitted through germ cells to the next generation as fully heritable mutations. This represents a molecular‑domain solution to the principal barrier limiting prime editing in plants.

Establishing a standard for multiplexed plant genome engineering and climate‑resilient crop design The impact of this genome‑editing engineering data on plant synthetic biology and green‑bio R&D is decisive because it establishes a commercial standard for ‘non‑toxic multiplex prime editing.’ Implementing complex traits such as increased yield or drought/pest resistance requires simultaneous precise editing of dozens of genomic sites; ‘Uncleavable Csy4‑PE’ enables this without cell death, acting as a safe insulating layer. It will serve as a unique hardware foundation for future plant‑specific guide‑design algorithms and AI‑driven editor‑optimization engines (e.g., BioArx green‑bio layers).