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.

中文翻译：

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通过结构建模对锌指核酸酶进行工程改造可提高细胞中的基因组编辑效率

基因组编辑广泛应用于生物医学研究和医学。锌指核酸酶 (ZFN) 的大小比转录激活因子样效应物 (TALE) 核酸酶 (TALEN) 和 CRISPR-Cas9 小。因此，ZFN编码DNA可以很容易地包装到载货空间有限的病毒载体中，例如腺相关病毒（AAV）载体，用于体内和临床应用。 ZFN 在转化研究和临床应用方面具有巨大潜力。然而，构建功能性ZFN并提高其基因组编辑效率极其困难。在此，描述了使用 AlphaFold、Coot 和 Rosetta 有效构建功能性 ZFN 以及提高其基因组编辑效率。使用公开可用的锌指资源组装编码由六个指组成的 ZFN 的质粒。从测试的 10 个 ZFN 中成功获得了两个功能 ZFN。此外，使用AlphaFold、Coot或Rosetta对ZFN进行工程化使基因组编辑效率提高了5%，证明了基于结构建模的ZFNs工程化的有效性。