The challenges of engineering zinc-finger nucleases

A protein containing three zinc fingers in complex with DNA. The zinc ions are highlighted in green.

Artificial zinc-finger DNA-binding proteins (ZFPs) have been engineered for quite some time now, and can be very useful in molecular biology. Zinc fingers are small protein structural motifs that can coordinate one or more zinc ions to help stabilize their folds. These structural motifs are involved in a broad range of biological activities including DNA binding, DNA and RNA recognition, as well as coordinating protein-protein interactions. One use of ZFPs is the creation of a sequence-specific nuclease, which can be used, for example, to mutate chromosomal targets, via a double-stranded DNA break, or to integrate foreign DNA into a locus. These are called zinc finger nucleases (ZFNs).

Even though a multitude of methods exist for engineering zinc fingers, making ZFNs is not a solved problem. Mark Isalan, head of the Gene Network Engineering group at the CRG, has recently written a commentary in Nature Methods on the issues faced when engineering these nucleases. This follows the group’s recent work on making zinc finger nucleases to repair p53 – the gene most commonly mutated in human cancer.

The Zinc Finger Consortium provides convenient computational tools to help design constructs, although this can still be challenging for inexperienced users, warns Isalan.

One of the reasons that ZFNs are challenging to build is that it is not possible to target just any desired DNA sequence. Most of the zinc fingers engineered have G-rich consensus sequences, because G-rich sequences are the natural preference of zinc fingers. Assembly of fingers for other types of targets is frequently unsuccessful.

The second difficulty is that in order to engineer a nuclease one needs to put together two domains (it’s a dimer) and it is much easier to make a single designer DNA-binding domain than to join together two domains, in the appropriate orientation, with the correct spacing, etc.

Isalan comments about these and other problems, and talks about some alternative new technologies, such as meganucleases and transcription activator–like effector (TALE) nucleases.

Reference:

Isalan M (2011). Zinc-finger nucleases: how to play two good hands. Nature methods, 9 (1), 32-4 PMID: 22205514

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