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:

1. Enhanced Prime Editing Efficiency with uPEn: The upgraded nuclease prime editor (uPEn) incorporates a ubiquitin variant (i53) to enhance double-strand break (DSB) repair, leading to a significant increase in gene-editing efficiency. Compared to traditional prime editors, uPEn improves knock-in rates while maintaining high precision, making it a promising tool for precise genome modifications.
2. Precise Genetic Modifications in Mouse Models: Using uPEn, researchers successfully inserted a consensus Kozak sequence into the PPARG (γ2) gene in mouse embryos, resulting in increased PPARγ2 protein expression in adipocytes. Next-generation sequencing (NGS) analysis confirmed that the modifications were highly accurate, with minimal unintended mutations, demonstrating the platform’s effectiveness in precise gene editing.
3. Successful Multiplexed Editing in Sheep Zygotes: The study applied uPEn to Hu sheep zygotes, achieving simultaneous PPARG (γ2) knock-in and MSTN knockout with high efficiency. A significant proportion of newborn lambs exhibited successful gene modifications, and some MSTN-knockout lambs displayed muscle hypertrophy, confirming that the genetic edits led to the expected physiological changes.
4. Efficient Germline Transmission of Edited Alleles: Founder animals carrying the edited PPARG (γ2) and MSTN genes successfully transmitted the modifications to their F1 offspring, demonstrating that the genetic changes were stably inherited. In mice, the PPARG (γ2) knock-in resulted in increased PPARγ2 protein expression in adipocytes, confirming the functional relevance of the modification and highlighting its potential applications in livestock breeding and metabolic research.

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).
DOI：10.1093/procel/pwaf006