Precision Genome Editing using Modulators of dsDNA Break Repair Pathways

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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.