Genome Editing Innovation Center, Hiroshima University, Higashi-Hiroshima, 739-0046, Japan.
Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Higashi-Hiroshima, 739-0046, Japan.
Adv Sci (Weinh). 2024 Jun;11(23):e2310255. doi: 10.1002/advs.202310255. Epub 2024 Apr 10.
Genome Editing is widely used in biomedical research and medicine. Zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector (TALE) nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFN-encoding DNAs can be easily packaged into a viral vector with limited cargo space, such as adeno-associated virus (AAV) vectors, for in vivo and clinical applications. ZFNs have great potential for translational research and clinical use. However, constructing functional ZFNs and improving their genome editing efficiency is extremely difficult. Here, the efficient construction of functional ZFNs and the improvement of their genome editing efficiency using AlphaFold, Coot, and Rosetta are described. Plasmids encoding ZFNs consisting of six fingers using publicly available zinc-finger resources are assembled. Two functional ZFNs from the ten ZFNs tested are successfully obtained. Furthermore, the engineering of ZFNs using AlphaFold, Coot, or Rosetta increases the efficiency of genome editing by 5%, demonstrating the effectiveness of engineering ZFNs based on structural modeling.
基因组编辑在生物医学研究和医学中得到了广泛应用。锌指核酸酶（ZFNs）比转录激活因子样效应物（TALENs）和 CRISPR-Cas9 更小。因此，ZFN 编码 DNA 可以很容易地包装到有限载量的病毒载体中，如腺相关病毒（AAV）载体，用于体内和临床应用。ZFN 具有转化研究和临床应用的巨大潜力。然而，构建功能性 ZFN 并提高其基因组编辑效率非常困难。在这里，描述了使用 AlphaFold、Coot 和 Rosetta 来高效构建功能性 ZFN 并提高其基因组编辑效率。组装了使用公共锌指资源的由六个手指组成的 ZFN 编码质粒。在测试的十个 ZFN 中，成功获得了两个功能性 ZFN。此外，使用 AlphaFold、Coot 或 Rosetta 对 ZFN 进行工程改造可将基因组编辑效率提高 5%，表明基于结构建模的 ZFN 工程改造是有效的。
