Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells

• Published in: Nucleic acids research 2005-01, Vol.33 (18), p.5978-5990
• Main Authors: Durai, Sundar, Mani, Mala, Kandavelou, Karthikeyan, Wu, Joy, Porteus, Matthew H., Chandrasegaran, Srinivasan
• Format: Article
• Language: English
• Subjects: Animals; Catalytic Domain; Deoxyribonucleases, Type II Site-Specific - chemistry; DNA Repair; DNA-Binding Proteins - chemistry; Gene Targeting; Genetic Engineering; Genome, Human; Genome, Plant; Genomics; Humans; Survey and Summary; Zinc Fingers
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gki912

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.