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, 31 (7), 397-405

ZFN, TALEN, and CRISPR/Cas-based Methods for Genome Engineering


ZFN, TALEN, and CRISPR/Cas-based Methods for Genome Engineering

Thomas Gaj et al. Trends Biotechnol.


Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases.


Figure 1
Figure 1. Structure of zinc-finger and transcription activator-like effectors
(a) (Top) Designed zinc-finger protein in complex with target DNA (grey) (PDB ID: 2I13). Each zinc-finger consists of approximately 30 amino acids in an ββα arrangement (inset). Surface residues (−1, 2, 3 and 6) that contact DNA are shown as sticks. Each zinc-finger domain contacts 3–4 base pairs (bps) in the major groove of DNA. The side chains of the conserved Cys and His residues are depicted as sticks in complex with a Zn2+ ion (purple). (b) Cartoon of a zinc-finger nuclease (ZFN) dimer bound to DNA. ZFN target sites consist of two zinc-finger binding sites separated by a 5- to 7-bp spacer sequence recognized by the FokI cleavage domain. Zinc-finger proteins can be designed to recognize unique “left” and “right” half-sites. (c) (Top) TALE protein in complex with target DNA (grey) (PDB ID: 3UGM). Individual TALE repeats contain 33–35 amino acids that recognize a single bp via two hypervariable residues (repeat-variable diresidues; RVDs) (shown as sticks) (inset). (d) Cartoon of a TALE nuclease (TALEN) dimer bound to DNA. TALEN target sites consist of two TALE binding sites separated by a spacer sequence of varying length (12-to 20-bp). TALEs can be designed to recognize unique “left” and “right” half-sites. RVD compositions are indicated.
Figure 2
Figure 2. Overview of possible genome editing outcomes using site-specific nucleases
Nuclease-induced DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or error-prone non-homologous end joining (NHEJ). (a) In the presence of donor plasmid with extended homology arms, HDR can lead to the introduction of single or multiple transgenes to correct or replace existing genes. (b) In the absence of donor plasmid, NHEJ-mediated repair yields small insertion or deletion mutations at the target that cause gene disruption. In the presence of double-stranded oligonucleotides or in vivo linearized donor plasmid, DNA fragments up to 14 kb have been inserted via NHEJ-mediated ligation. Simultaneous induction of two DSBs can lead to deletions, inversions and translocations of the intervening segment.

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