Precision Genome Editing using Modulators of dsDNA Break Repair Pathways

使用 dsDNA 断裂修复途径调节剂进行精准基因组编辑

• 批准号: 2429439
• 金额: --
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2429439
• 关键词: Precision; Genome; Editing; using; Modulators

CRISPR/Cas9 relies on introducing specific double-strand breaks into the target genome which are then repaired by Homology-Directed Repair (HDR) or Non-Homologous End-Joining (NHEJ); the activity of these pathways depends on the type and dividing state of the cell. Both DNA repair mechanisms can be utilized productively for DNA insertion into the genome, each with its own merits. However, in the absence of any selection pressure, the DNA insertion efficiency is low (<10% typically) preventing exploitation of CRISPR/Cas9 systems for large DNA insertions to their full potential.We aim to potentiate the efficacy of DNA integration into the target genome. Chemicals have been shown to enhance DNA repair by selectively modulating components of the cellular DNA repair machinery. Therefore, we postulate that nanobodies can likewise act on the cellular DNA repair factors, inhibiting either HDR or NHEJ and thus boosting efficacies. Nanobodies (Camelid single-domain antibody fragments) are small (15 kDa), and fold within cells. In this project we will start from a naïve nanobody library and select highly specific, high-affinity binders using Ribosome Display in vitro selection and evolution, a method established in the Berger-Schaffitzel laboratory. Many constituent proteins of HDR and NHEJ are already available in recombinant, purified form in Mark Dillingham's lab; these will be used as antigens for nanobody selection. We have already shown that this is possible by selecting nanobodies against KU70/80. DNA encoding for the best nanobody candidates with desired effects (such as blocking protein-protein interactions, inhibiting Ku70/80 DNA binding, etc) will be incorporated into viral vectors that already contain the CRISPR/Cas9 system to boost target-DNA insertion efficacy. To this end, the selected nanobody binders will be characterized biochemically, biophysically and structurally (using crystallography or electron cryo-microscopy). In addition, in collaboration with Dr Binyam Mogessie, we will establish Trim-Away for those nanobodies which bind but do not interfere with the target's function. Trim-Away is a highly effective technique to acutely degrade endogenous proteins in mammalian cells with the help of antibodies. Antibody-bound targets are recognized by TRIM21 that recognizes the Fc domain of antibodies with high affinity. Therefore, we will fuse the human Fc domain to our selected nanobody. TRIM21 will then target the nanobody-antigen complex (e.g. a protein of the dsDNA repair pathway) to the proteasome for degradation. This approach offers exciting possibilities for acute, transient protein depletion (within minutes of application, avoiding secondary, compensatory effects) to study basic mechanisms of dsDNA repair and boost DNA insertion efficiency.

CRISPR/Cas9 依赖于在目标基因组中引入特定的双链断裂，然后通过同源定向修复 (HDR) 或非同源末端连接 (NHEJ) 进行修复；这些途径的活性取决于细胞的类型和分裂状态。这两种 DNA 修复机制都可以有效地用于将 DNA 插入基因组，每种机制都有自己的优点。然而，在没有任何选择压力的情况下，DNA 插入效率较低（通常<10%），阻碍了利用 CRISPR/Cas9 系统进行大型 DNA 插入的充分潜力。我们的目标是增强 DNA 整合到目标基因组中的效率。化学物质已被证明可以通过选择性调节细胞 DNA 修复机制的成分来增强 DNA 修复。因此，我们假设纳米抗体同样可以作用于细胞 DNA 修复因子，抑制 HDR 或 NHEJ，从而提高疗效。纳米抗体（骆驼单域抗体片段）很小（15 kDa），并且在细胞内折叠。在这个项目中，我们将从一个原始的纳米抗体库开始，并使用核糖体展示体外选择和进化（一种在 Berger-Schaffitzel 实验室建立的方法）来选择高度特异性、高亲和力的结合物。 Mark Dillingham 的实验室已提供重组、纯化形式的 HDR 和 NHEJ 的许多组成蛋白；这些将用作纳米抗体选择的抗原。我们已经证明，通过选择针对 KU70/80 的纳米抗体，这是可能的。编码具有所需效果（例如阻断蛋白质-蛋白质相互作用、抑制 Ku70/80 DNA 结合等）的最佳纳米抗体候选物的 DNA 将被整合到已包含 CRISPR/Cas9 系统的病毒载体中，以提高靶标 DNA 插入效率。为此，将对所选纳米抗体结合剂进行生物化学、生物物理和结构表征（使用晶体学或电子冷冻显微镜）。此外，我们将与 Binyam Mogessie 博士合作，为那些结合但不干扰靶标功能的纳米抗体建立 Trim-Away。 Trim-Away 是一种在抗体的帮助下急剧降解哺乳动物细胞内源蛋白的高效技术。 TRIM21 可以识别抗体结合的靶标，TRIM21 能够以高亲和力识别抗体的 Fc 结构域。因此，我们将把人类 Fc 结构域融合到我们选择的纳米抗体上。然后 TRIM21 会将纳米抗体-抗原复合物（例如 dsDNA 修复途径的蛋白质）靶向蛋白酶体进行降解。这种方法为急性、短暂的蛋白质消耗（应用后几分钟内，避免继发性补偿效应）提供了令人兴奋的可能性，以研究 dsDNA 修复的基本机制并提高 DNA 插入效率。