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Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
Published on: January 3, 2015
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Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9
Luqing Zhang1, Ruirui Jia2, Norberto J Palange2
1Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China; Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast Normal University, Changchun, China.
Plos One
|March 25, 2015
Summary
The CRISPR/Cas9 gene editing system efficiently creates large DNA deletions and insertions in mice, enabling new mouse models for gene function studies. This method is fast, cost-effective, and simplifies genetic engineering.
Area of Science:
- Genomic engineering and molecular biology.
- Development of advanced gene editing tools.
Background:
- CRISPR/Cas9, ZFNs, and TALENs are gene editing tools for genomic modifications.
- CRISPR/Cas9 is a flexible technology widely used across organisms.
- Previous CRISPR/Cas9 applications primarily focused on small insertions and deletions.
Purpose of the Study:
- To demonstrate the CRISPR/Cas9 system's capability for large DNA fragment deletions and insertions in mice.
- To establish efficient methods for generating large genomic modifications using CRISPR/Cas9.
- To facilitate gene function studies through rapid and cost-effective mouse model generation.
Main Methods:
- Utilized CRISPR/Cas9 system for large DNA fragment manipulation in mouse embryonic stem (ES) cells and zygotes.
- Performed targeted deletion of the entire Dip2a gene (approx. 65kb) and insertion of a β-galactosidase (lacZ) reporter gene (over 5kb).
- Employed direct co-injection of zygotes or co-transfection of ES cells with circular plasmid DNA, bypassing in vitro transcription.
Main Results:
- Achieved 11.8% positive rate for 65kb deletion in ES clones and high targeting efficiencies (46.2-73.1%) with G418 selection.
- Demonstrated targeted large fragment deletion efficiency of 21.4% in live pups and 6.0% in injected embryos.
- Obtained targeting rates of 27.1% for lacZ insertion via ES cell transfection and 11.1% via direct zygote injection.
Conclusions:
- CRISPR/Cas9 system effectively generates large DNA deletions and insertions in mouse models.
- The described methods are technically straightforward, time-saving, and cost-effective.
- These advancements significantly facilitate the study of gene function through improved mouse model generation.
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