The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA, Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA and Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Nucleic Acids Res. 2014 Apr;42(7):4755-66. doi: 10.1093/nar/gkt1389. Epub 2014 Jan 21.
The serine recombinases are a diverse family of modular enzymes that promote high-fidelity DNA rearrangements between specific target sites. Replacement of their native DNA-binding domains with custom-designed Cys₂-His₂ zinc-finger proteins results in the creation of engineered zinc-finger recombinases (ZFRs) capable of achieving targeted genetic modifications. The flexibility afforded by zinc-finger domains enables the design of hybrid recombinases that recognize a wide variety of potential target sites; however, this technology remains constrained by the strict recognition specificities imposed by the ZFR catalytic domains. In particular, the ability to fully reprogram serine recombinase catalytic specificity has been impeded by conserved base requirements within each recombinase target site and an incomplete understanding of the factors governing DNA recognition. Here we describe an approach to complement the targeting capacity of ZFRs. Using directed evolution, we isolated mutants of the β and Sin recombinases that specifically recognize target sites previously outside the scope of ZFRs. Additionally, we developed a genetic screen to determine the specific base requirements for site-specific recombination and showed that specificity profiling enables the discovery of unique genomic ZFR substrates. Finally, we conducted an extensive and family-wide mutational analysis of the serine recombinase DNA-binding arm region and uncovered a diverse network of residues that confer target specificity. These results demonstrate that the ZFR repertoire is extensible and highlights the potential of ZFRs as a class of flexible tools for targeted genome engineering.
丝氨酸重组酶是一类具有多样性的模块化酶，可在特定靶位之间促进高保真度的 DNA 重排。用自定义设计的 Cys₂-His₂锌指蛋白替换其天然的 DNA 结合结构域，会产生能够实现靶向基因修饰的工程化锌指重组酶（ZFR）。锌指结构域的灵活性使设计能够识别各种潜在靶位的杂交重组酶成为可能；然而，这项技术仍然受到 ZFR 催化结构域严格识别特异性的限制。具体来说，由于每个重组酶靶位中都存在保守的碱基要求，以及对决定 DNA 识别的因素缺乏完整的了解，完全重新编程丝氨酸重组酶催化特异性的能力受到了阻碍。在这里，我们描述了一种补充 ZFR 靶向能力的方法。通过定向进化，我们分离到了 β 和 Sin 重组酶的突变体，这些突变体能特异性识别以前不在 ZFR 范围内的靶位。此外，我们开发了一种遗传筛选方法来确定位点特异性重组的特定碱基要求，并表明特异性分析能够发现独特的基因组 ZFR 底物。最后，我们对丝氨酸重组酶 DNA 结合臂区域进行了广泛的家族突变分析，揭示了赋予靶位特异性的多样化残基网络。这些结果表明 ZFR 库是可扩展的，并强调了 ZFR 作为一类用于靶向基因组工程的灵活工具的潜力。
