Precision Genome Editing using Modulators of dsDNA Break Repair Pathways

CRISPR/Cas9依赖于将特定的双链断裂引入目标基因组，然后通过同源定向修复（HDR）或非同源末端连接（NHEJ）进行修复；这些通路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插入效率很低（通常<10%），阻碍了CRISPR/Cas9系统对大DNA插入的充分利用。我们的目标是增强DNA整合到目标基因组中的功效。化学物质已被证明可以通过选择性地调节细胞DNA修复机制的组成部分来增强DNA修复。因此，我们假设纳米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。纳米小体（Camelid single-domain antibody fragments）很小（15 kDa），在细胞内折叠。在这个项目中，我们将从naïve纳米体文库开始，使用在Berger-Schaffitzel实验室建立的方法，在体外选择和进化中使用核糖体展示来选择高特异性、高亲和力的结合物。在Mark Dillingham的实验室里，HDR和NHEJ的许多组成蛋白已经以重组和纯化的形式存在；这些将被用作纳米体选择的抗原。我们已经证明，通过选择抗KU70/80的纳米体，这是可能的。编码具有理想效果（如阻断蛋白-蛋白相互作用、抑制Ku70/80 DNA结合等）的最佳候选纳米体的DNA将被整合到已经包含CRISPR/Cas9系统的病毒载体中，以提高目标DNA插入效率。为此，所选择的纳米体粘合剂将被生物化学、生物物理和结构（使用晶体学或电子冷冻显微镜）表征。此外，在与Binyam Mogessie博士的合作下，我们将为那些结合但不干扰目标功能的纳米体建立Trim-Away。Trim-Away是一种非常有效的技术，可以在抗体的帮助下快速降解哺乳动物细胞中的内源性蛋白质。抗体结合的靶标被TRIM21识别，它识别高亲和力抗体的Fc结构域。因此，我们将人类Fc域融合到我们选择的纳米体中。然后TRIM21将纳米体抗原复合物（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、瞬时蛋白质耗竭（在几分钟内应用，避免继发性补偿效应）提供了令人兴奋的可能性，以研究dsDNA修复的基本机制和提高DNA插入效率。

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.