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，其原则上可用于通过同源重组修复基因。这种新兴技术受到其低效率的限制;然而，通过在特定基因座内进行特定的双链断裂，它可以增加1000倍以上。用于此目的的主要工具是归巢核酸内切酶。这些酶识别长度高达40个碱基对的DNA序列，并仅在少数位置切割大基因组。对于特异性基因靶向，期望具有预定特异性的归巢核酸内切酶。我们建议通过定向进化来创建这样的酶，利用一些归巢核酸内切酶具有远离催化中心的识别模块的事实。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核酸内切酶是否具有足够的结构可塑性，使得有限数量的氨基酸取代可以产生特定的特异性变化。因此，我们的项目将导致更好地理解DNA识别的约束条件的预制结构元件。

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.