Generation of homing endonucleases of pre-defined specificity by directed evolution

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A major goal in the post-genomic era is to develop tools for gene targeting which in principle could be used to repair genes via homologous recombination. This emerging technology is limited by its low efficiency; however it can be increased over 1000-fold by making a specific double strand break within a particular locus. Prime tools for this purpose are homing endonucleases. These enzymes recognize DNA sequences of up to 40 base pairs in length and cleave large genomes only at a few positions. For specific gene targeting it would be desirable to have homing endonucleases with pre-defined specificity. We propose to create such enzymes by directed evolution, making use of the fact that some homing endonucleases have recognition modules distal from the catalytic center. This should allow manipulation of the DNA binding module without affecting the catalytic activity. Our study will determine whether homing endonucleases are sufficiently structurally malleable, such that a limited number of amino acid substitutions can produce a defined change in specificity. Thus, our project will lead to a better understanding of the constraints for DNA recognition by preformed structural elements.