Cell-free protein-evolution systems for engineering of novel sequence-specific dna-binding and -modifying activities

Engineered DNA-binding proteins, in particular zinc finger proteins (ZFPs), comprise a promising new class of therapeutic proteins for applications in gene therapy that range from regulation of gene expression to in vivo mutation correction. All these approaches depend on the availability of highly specific DNA-binding proteins for genome-wide accurate targeting of therapeutic constructs. Until now, the design of novel ZFPs has mainly been achieved by selection using phage display. This is a labor-intensive approach which allows working with only moderatelysized combinatorial libraries. We overcame these limitations by adapting a convenient cell-free selection method known as in vitro compartmentalization (IVC) for the selection of ZFPs. In IVC, the expression libraries are assembled by PCR and up to 1010 independent clones can be selected conveniently in a single reaction. In addition to ZFPs, IVC has previously been used to engineer DNA-modifying enzymes, e.g., polymerase and methyltransferase activities. In this chapter, we describe the IVC procedure and review the progress made.