Targeted gene knockout by direct delivery of zinc-finger nuclease proteins

• Published in: Nature methods Vol. 9; no. 8; pp. 805 - 807
• Main Authors: Gaj, Thomas, Guo, Jing, Kato, Yoshio, Sirk, Shannon J, Barbas, Carlos F
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.08.2012; Nature Publishing Group
• Subjects: 631/1647/1513/1967/2315; 631/1647/338; 631/61/2297; Amino Acid Motifs; Amino Acid Sequence; Animals; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; brief-communication; Catalytic Domain; Cell Membrane Permeability; Cells; CHO Cells; Cricetinae; Deoxyribonucleases, Type II Site-Specific - chemistry; Deoxyribonucleases, Type II Site-Specific - metabolism; Disruption; Gene disruption; Gene Knockout Techniques - methods; Gene therapy; Genes; Genetic aspects; Genetic engineering; Genetic Engineering - methods; Genome editing; Genomes; Genomics; Humans; Life Sciences; Mammals; Molecular Sequence Data; Nuclease; Nucleases; Peptides - chemistry; Physiological aspects; Proteins; Proteomics; Reagents; Receptors, CCR5 - genetics; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; tat Gene Products, Human Immunodeficiency Virus - chemistry; Zinc; Zinc finger proteins; Zinc Fingers - genetics; Zinc Fingers - physiology; United States
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth.2030

Due to an unexpected cell-penetrating property, zinc-finger nucleases (ZFNs) can be delivered to several mammalian cell types as proteins. Dose-dependent disruption of an endogenous gene was achieved with reduced activity at known off-target sites. Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in various mammalian cell types with minimal off-target effects.