Chimeric restriction enzymes are a novel class of engineered nucleases in which the nonspecific DNA cleavage domain (a type IIs restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely, the zinc finger motif, the helix-turn-helix motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by micro-injection of DNA substrates and the enzyme into frog oocytes. The injected enzyme made site-specific double-strand breaks in the targets even after assembly of DNA into chromatin. In addition, this cleavage activated the target modules for efficient homologous recombination. The goal here is to redesign and improve the chimeric nucleases by computer modeling based on the crystal structures of native FokI and FokI bound to DNA. The investigators also plan to increase the sequence specificity of the hybrid endonucleases by using polydactyl zinc fingers. They plan to induce homologous recombination at a chromosomal site by using engineered chimeric nucleases within a human or animal cell line. Their long term goal is the application of chimeric nucleases to correct a genetic defect in an animal model.
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