CRISPR/Cas9 has revolutionized genome editing, but prime editing (PE) offers a more precise, DSB-independent approach. However, PE efficiency remains suboptimal, especially for large animal genetic modifications. To address this, researchers developed uPEn, integrating a ubiquitin variant (i53) to enhance genome stability and repair efficiency.
The study applied uPEn to insert a consensus Kozak sequence into PPARG (γ2), a gene linked to fat deposition, in mouse and sheep zygotes. Mouse trials demonstrated efficient Kozak motif insertion, with improved PPARγ2 expression in adipocytes, validating the strategy. In sheep, uPEn was used for dual-gene editing, achieving high-efficiency PPARG knock-in and MSTN knockout, a gene regulating muscle growth.
NGS analyses confirmed precise modifications with minimal off-target effects. Furthermore, F0 founder animals successfully transmitted edited alleles to offspring. These results suggest uPEn as a versatile and scalable genome-editing platform for livestock improvement and biomedical research.
Key findings from the study include:
This study introduces uPEn, an optimized nuclease prime editor that significantly enhances the efficiency and precision of genetic modifications in mammalian embryos. The successful PPARG (γ2) Kozak motif insertion in mice demonstrated the platform’s capability for precise knock-ins. Furthermore, the dual-gene editing of PPARG and MSTN in Hu sheep highlights its potential for complex genetic modifications.
The results confirm that uPEn enables highly efficient, multiplexed genome engineering in livestock with minimal off-target effects, paving the way for agricultural improvements, disease modeling, and gene therapy applications. Future research will refine this technology by integrating high-fidelity Cas9 variants and further optimizing RNA designs to enhance editing efficiency. The work entitled “ An Upgraded Nuclease Prime Editor Platform Enables High-Efficiency Singled or Multiplexed Knock-In/Knockout of Genes in Mouse and Sheep Zygotes ” was published on Protein & Cell (published on Jan. 20, 2025).
Protein & Cell
Experimental study
Animals
An upgraded nuclease prime editor platform enables high-efficiency singled or multiplexed knock-in/knockout of genes in mouse and sheep zygotes
20-Jan-2025