Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology, Saudi Arabia.
Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology, Saudi Arabia.
J Biol Chem. 2024 Sep;300(9):107720. doi: 10.1016/j.jbc.2024.107720. Epub 2024 Aug 28.
Site-specific nucleases are crucial for genome engineering applications in medicine and agriculture. The ideal site-specific nucleases are easily reprogrammable, highly specific in target site recognition, and robust in nuclease activities. Prokaryotic Argonaute (pAgo) proteins have received much attention as biotechnological tools due to their ability to recognize specific target sequences without a protospacer adjacent motif, but their lack of intrinsic dsDNA unwinding activity limits their utility in key applications such as gene editing. Recently, we developed a pAgo-based system for site-specific DNA cleavage at physiological temperatures independently of the DNA form, using peptide nucleic acids (PNAs) to facilitate unwinding dsDNA targets. Here, we fused catalytically dead pAgos with the nuclease domain of the restriction endonuclease FokI and named this modified platform PNA-assisted FokI-(d)pAgo (PNFP) editors. In the PNFP system, catalytically inactive pAgo recognizes and binds to a specific target DNA sequence based on a programmable guide DNA sequence; upon binding to the target site, the FokI domains dimerize and introduce precise dsDNA breaks. We explored key parameters of the PNFP system including the requirements of PNA and guide DNAs, the specificity of PNA and guide DNA on target cleavage, the optimal concentration of different components, reaction time for invasion and cleavage, and ideal temperature and reaction buffer, to ensure efficient DNA editing in vitro. The results demonstrated robust site-specific target cleavage by PNFP system at optimal conditions in vitro. We envision that the PNFP system will provide higher editing efficiency and specificity with fewer off-target effects in vivo.
在医学和农业领域的基因组工程应用中，位点特异性核酸酶至关重要。理想的位点特异性核酸酶易于重新编程，在靶序列识别方面具有高度特异性，并且在核酸酶活性方面稳健。由于能够在没有前导序列相邻基序的情况下识别特定的靶序列，原核 Argonaute (pAgo) 蛋白作为生物技术工具受到了广泛关注，但由于其缺乏内在的双链 DNA 解旋活性，限制了其在关键应用中的使用，例如基因编辑。最近，我们开发了一种基于 pAgo 的系统，可在生理温度下独立于 DNA 形式进行特异性 DNA 切割，使用肽核酸 (PNA) 促进双链 DNA 靶标的解旋。在这里，我们将无活性的 pAgos 与限制性内切酶 FokI 的核酸酶结构域融合，并将这个经过修饰的平台命名为 PNA 辅助 FokI-(d)pAgo (PNFP) 编辑器。在 PNFP 系统中，无活性的 pAgo 基于可编程的向导 DNA 序列识别和结合特定的靶 DNA 序列；与靶位点结合后，FokI 结构域二聚化并引入精确的双链 DNA 断裂。我们探索了 PNFP 系统的关键参数，包括 PNA 和向导 DNA 的要求、PNA 和向导 DNA 对靶切割的特异性、不同成分的最佳浓度、入侵和切割的反应时间以及理想的温度和反应缓冲液，以确保在体外进行有效的 DNA 编辑。结果表明，PNFP 系统在体外优化条件下可实现稳健的特异性靶标切割。我们设想，PNFP 系统将在体内提供更高的编辑效率和特异性，同时减少脱靶效应。
