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Updated: Jun 23, 2026
10:07
A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
Published on: August 25, 2017
Prime editing-mediated microhomology enables efficient replacement of large DNA
Yuyang Xie1, Pan Li2, Zhiyong He1
1Frontiers Science Center for Molecular Design Breeding (MOE), State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Nucleic Acids Research
|June 22, 2026
Summary
Prime Editing-Microhomology-Enabled Replacement (PREMIER) is a new genome engineering tool that precisely replaces large DNA segments without causing double-strand breaks. This DSB-free method achieves high efficiency for large-scale genome rewriting and therapeutic applications.
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- Precise large genomic DNA segment replacement without double-strand breaks (DSBs) is a major genome engineering challenge.
- Existing methods like homologous recombination and prime editing (PE) have limitations, including inefficiency, reliance on DSBs, and difficulty with large DNA fragments.
Purpose of the Study:
- To develop a novel, DSB-free genome engineering platform for efficient and precise replacement of large genomic DNA segments.
- To overcome the limitations of current prime editing techniques in integrating large DNA fragments.
Main Methods:
- Introduction of Prime Editing-Microhomology-Enabled Replacement (PREMIER), a DSB-free platform.
- Utilizing PE to install single-stranded microhomology arms at donor and genomic junctions.
- Testing PREMIER in cell lines and in vivo models for large DNA fragment integration and gene replacement.
Main Results:
- PREMIER demonstrated high efficiency (mean 63.4%, peak 85.9%) in diverse cell line targets, outperforming homology-directed repair 10-20 fold.
- Reduced off-target integrations by over 100-fold compared to nonhomologous end joining.
- Successfully integrated large DNA fragments up to 10.3 kb in vitro and a 6.2-kb oncogene cassette in vivo, and generated humanized mice by replacing Trp53 with human TP53.
Conclusions:
- PREMIER offers a precise, highly efficient, and DSB-free strategy for large-scale genome rewriting.
- This platform simplifies donor preparation and bypasses the need for long homology arms.
- PREMIER is a powerful tool for complex genome modeling and therapeutic genome editing applications.
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