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Genome editing with engineered zinc finger nucleases

Nature Reviews Genetics volume 11, pages 636–646 (2010)Cite this article

Subjects

Key Points

  • Targeted genetic engineering in many important model systems and in human tissue culture cells has historically been challenging. This has changed dramatically over the past 5 years with the development of zinc finger nuclease (ZFN) technology.

  • A ZFN is an artificial endonuclease that consists of a designed zinc finger protein (ZFP) fused to the cleavage domain of the FokI restriction enzyme. A ZFN may be redesigned to cleave new targets by developing ZFPs with new sequence specificities.

  • For genome engineering, a ZFN is targeted to cleave a chosen genomic sequence. The cleavage event induced by the ZFN provokes cellular repair processes that in turn mediate efficient modification of the targeted locus.

  • If the ZFN-induced cleavage event is resolved via non-homologous end joining, this can result in small deletions or insertions, effectively leading to gene knockout. This approach has now been used to establish facile and efficient reverse genetics (that is, reverse genetics that does not require selection) in Drosophila melanogaster, zebrafish, rats, Arabidopsis thaliana and mammalian somatic cells.

  • If the break is resolved via a homology-based process in the presence of an investigator-provided donor, small changes or entire transgenes can be transferred, often without selection, into the chromosome; this is referred to as 'gene correction' and 'gene addition', respectively. This approach has been used to make novel alleles in D. melanogaster, mammalian cells and tobacco, and has been used to drive targeted integration in maize, tobacco and human embryonic stem and induced pluripotent stem cells.

  • Therapeutic application of ZFN technology requires the engineering of ZFNs that are highly specific in their action. Three clinical trials with ZFNs are underway, including one in which T cells are isolated from a patient infected with HIV, treated with ZFNs that disrupt the chemokine (C-C motif) receptor type 5 (CCR5) gene to make them resistant to virus infection, and transferred back to the patient.

Abstract

Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells — this broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a number of model organisms, thus opening the door to a range of new experimental and therapeutic possibilities.

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Figure 1: Structure and design of zinc finger nucleases.
Figure 2: Eliminating potential homodimerization.
Figure 3: Types of genome editing made possible using zinc finger nucleases.

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  1. Sangamo BioSciences Inc., 501 Canal Boulevard, Suite A100, Richmond, 94804, California, USA

    Fyodor D. Urnov, Edward J. Rebar, Michael C. Holmes, H. Steve Zhang & Philip D. Gregory

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Glossary

Paralogues

Two or more genes within a given species that originated from a single parent gene via duplication events, usually with a subsequent, sometimes subtle, divergence of function.

Pseudogenes

Non-functional paralogues that often lack promoter or intron sequences.

Adeno-associated viruses

(AAVs). As defined by Flint and colleagues in Principles of Virology, AAV is a parvovirus (also known as a dependovirus) that can establish a latent infection during which its DNA is integrated into the host cell genome in an inactive state. AAV2 integrates into the PPP1R12C gene locus on chromosome 19.

Homing endonuclease

A 'meganuclease', such as I-SceI, that recognizes and cuts longer sequences (18 bp in the case of I-SceI) than those cut by commonly used restriction enzymes.

Episomal

The formal, technical definition of 'episome', and its distinction from the word 'plasmid' (as proposed by Jacob and Wollman in 1958), is complex. In vernacular use it means 'circular extrachromosomal DNA molecule present inside the nucleus'.

Protoplasts

Plant cells that lack a cell wall.

Calli

(Sing. callus.) Clusters of undifferentiated plant cells grown on solid medium that, in some species and under specific culture conditions, have the capacity to regenerate a whole plant.

Short hairpin RNA

Small RNAs that form hairpins that can induce sequence-specific silencing in mammalian cells through RNAi.

Systematic evolution of ligands by exponential enrichment

(SELEX). A method for identifying nucleic acid ligands for a chosen 'bait' molecule (typically a protein). In its most general form, the method comprises: incubation of a randomized nucleic acid library with the bait molecule; recovery of the bait, along with any bound nucleic acids; amplification of recovered nucleic acids via PCR; and sequencing to identify binding motifs.

Embryogenic suspension cells

Plant cells, derived from callus tissue or from an embryo, that can be maintained in liquid growth medium and that, under appropriate culture conditions, can be used to regenerate a whole plant.

Autologous

In transplantology, referring to cells or an organ transplanted from an individual to that same individual (often after some ex vivo procedure has been performed).

Intrabodies

Antibodies that are directed against intracellular target molecules and expressed within a specific subcellular compartment as directed by localization signals genetically fused to the amino or carboxyl terminus of a given antibody.

Aviraemic

Aviraemia refers to the lack of detectable virus in the circulation of an individual.

Ultradeep sequencing

An umbrella term that refers to several independent, proprietary, high-throughput DNA-sequencing technologies that use massively parallel sequencing-by-synthesis approaches. The new methods allow an increase in generated sequence per run of about two orders of magnitude compared with conventional Sanger sequencing technologies.

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Urnov, F., Rebar, E., Holmes, M. et al. Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11, 636–646 (2010). https://doi.org/10.1038/nrg2842

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  • DOI: https://doi.org/10.1038/nrg2842

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