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Updated: Jul 6, 2026
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Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)
Published on: June 14, 2012
Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases
Yolanda Santiago1, Edmond Chan, Pei-Qi Liu
1Sangamo BioSciences, Inc., 501 Canal Boulevard, Suite A100, Richmond, CA 94804, USA.
Summary
This study introduces a rapid, single-step gene knockout method in mammalian cells using zinc-finger nucleases (ZFNs). This efficient technique enables targeted gene disruption for applications in functional genomics and drug discovery.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Gene knockout is crucial for understanding gene function and cell characteristics.
- Current gene disruption methods face limitations in efficiency, time, and off-target effects.
Purpose of the Study:
- To demonstrate a rapid, single-step gene knockout method in mammalian cells.
- To utilize engineered zinc-finger nucleases (ZFNs) for high-specificity gene targeting.
Main Methods:
- Designed ZFNs to target the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells.
- Transiently expressed ZFNs to induce targeted DNA cleavage, followed by nonhomologous end joining repair.
- Generated and characterized DHFR-knockout cell lines.
Main Results:
- Achieved biallelic DHFR gene disruption at frequencies greater than 1%, eliminating the need for selection markers.
- Successfully generated three distinct DHFR-knockout (DHFR-/-) CHO cell lines.
- Confirmed gene disruption within 2-3 days of ZFN delivery, with cell line isolation within one month.
Conclusions:
- ZFNs offer a powerful tool for rapid mammalian cell line engineering.
- This method provides a new approach for gene knockout applicable to reverse genetics, functional genomics, drug discovery, and biopharmaceutical production.
