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CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
Published on: March 12, 2018
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High-efficiency targeted transgene integration via primed micro-homologues
Chenxin Wang1,2,3,4, Sen Fang1,2,3, Yangcan Chen1,2,3
1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
Cell Discovery
|July 4, 2023
Summary
We developed PAINT 3.0, a prime editor-based gene editing tool that significantly improves large transgene knock-in (KI) efficiency and accuracy. This method offers a powerful new approach for gene therapies and genome engineering.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
- Synthetic Biology
Background:
- Precise manipulation of DNA repair pathways for large transgene integration remains a significant challenge.
- Current methods for targeted DNA integration often suffer from low efficiency and off-target effects.
Purpose of the Study:
- To develop a robust and efficient gene editing strategy for high-fidelity targeted integration of large transgenes.
- To enhance existing prime editor technology for improved knock-in (KI) efficiency and reduced off-target integration.
Main Methods:
- Exploited prime editors to create a novel knock-in (KI) strategy named primed micro-homologues-assisted integration (PAINT).
- Utilized reverse-transcribed single-stranded micro-homologues to facilitate targeted KIs.
- Developed PAINT 3.0, an improved version focusing on maximizing editing efficiency and minimizing off-target integration, particularly for scarless in-frame KIs.
Main Results:
- Achieved up to 80% editing efficiency when targeting a reporter transgene into housekeeping genes, exceeding traditional homology-directed repair by over 10-fold.
- Demonstrated up to 85% KI frequency for a 2.5-kb transgene at therapeutically relevant genomic loci.
- Enabled high-efficiency non-viral genome targeting in primary T cells, generating functional CAR-T cells with tumor-killing capabilities.
Conclusions:
- The PAINT method, particularly PAINT 3.0, is a powerful gene editing tool for large transgene integrations.
- This technology shows significant potential for advancing cell and gene therapies and genome writing.
- PAINT 3.0 offers a robust solution for efficient and precise genetic engineering applications.
