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

后基因组时代的一个主要目标是开发基因靶向工具，原则上可用于通过同源重组修复基因。这种新兴技术的局限性在于效率低；然而，通过在特定位点内制造特定的双链断裂，它可以增加1000倍以上。用于此目的的主要工具是归巢内切酶。这些酶可以识别长度达40个碱基对的DNA序列，并且只在少数位置切割大的基因组。对于特定的基因靶向，需要具有预定义特异性的归巢内切酶。我们建议通过定向进化来创造这样的酶，利用一些归巢内切酶在催化中心远端具有识别模块的事实。这应该允许在不影响催化活性的情况下操纵DNA结合模块。我们的研究将确定归巢内切酶是否具有足够的结构延展性，以便有限数量的氨基酸取代可以产生确定的特异性变化。因此，我们的项目将有助于更好地理解预先形成的结构元素对DNA识别的限制。

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.