Related Experiment Video
Updated: Jun 5, 2026
09:51
Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Published on: May 25, 2018
Optimizing microhomology-based genome editing by engineering DNA polymerase θ for improved efficiency and reduced
Wanyi Wang1, Meng Zhou2, Tianshan Ji3
1Department of Physiology, School of Basic Medical Science, Nanjing Medical University, Nanjing, Jiangsu 211166, China; Jiangsu Provincial Key Laboratory of Biological Therapy for Organ Failure, Nanjing Medical University, Nanjing, Jiangsu 211166, China.
Cell Reports Methods
|June 3, 2026
Summary
We developed a DNA polymerase theta (Pol θ)-based editor to improve precise genome editing, enhancing targeted DNA integration efficiency in various cell types for gene therapy applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Precise genome editing via targeted DNA insertion is crucial for gene therapy and research.
- Current homology-directed repair (HDR) methods are inefficient in non-dividing cells.
- Microhomology-mediated repair (MMEJ) is an alternative but also inefficient.
Purpose of the Study:
- To develop a novel DNA polymerase theta (Pol θ)-based editor (PET) for enhanced kilobase-scale targeted DNA integration.
- To improve the efficiency and precision of genome editing, particularly in challenging cell types.
Main Methods:
- Development of a Pol θ-based editor (PET) utilizing its polymerase (pPET) and helicase-like (hPET) domains.
- Evaluation of pPET and hPET for enhancing microhomology-mediated editing efficiency.
- Assessment of on-target insertion and indel rates using next-generation sequencing.
Main Results:
- pPET and hPET independently enhanced microhomology-mediated editing.
- pPET increased knock-in rates threefold compared to conventional SpCas9-mediated editing.
- pPET and hPET reduced on-target indel rates by approximately 30% and increased precise insertions by up to 80%.
Conclusions:
- Pol θ functional domains (pPET and hPET) are effective tools for improving microhomology-driven genome editing.
- PET technology offers a promising strategy for advancing therapeutic genome editing applications.
- This approach enhances precise DNA integration across diverse genomic loci and cell types.
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