Grant: Discovery Development Grant

BACKGROUND CRISPR/Cas genome editing is revolutionizing biomedical research. RNA-guided Cas9 nuclease can induce targeted double strand breaks (DSBs) in DNA, leading to knockout of the targeted locus by insertion or deletion of DNA (indels). Nevertheless, most applications of genome editing call for the introduction of specific mutations in the genome, rather than indels. This can be achieved by homology-directed repair (HDR) using a template DNA containing the desired mutation. However, the yields of HDR-mediated mutagenesis in current protocols are low, necessitating steps of selection/purification. GENERAL OBJECTIVE This research program aims at generating knowledge and methods toward achieving efficient, scarless genome modification. SPECIFIC OBJECTIVES AND METHODOLOGIES 1. To create a FACS-based system for monitoring functional gene modification in human cells. This will be achieved by the establishment of cellular models in which a surface protein, epidermal growth factor receptor (EGFR), is mutated by HDR, leading to detection by flow cytometry. Hence, the efficiency of HDR is reflected by the % of cells expressing the marker. 2. To optimize HDR-mediated modification. We will methodically compare the efficiencies of several types of donor (template) DNAs and delivery options in the context of integration-deficient lentiviral vectors. In particular, we will analyze their optimal length and orientation, and determine the optimal timing of delivery relative to the CRISPR/Cas components. 3. Improving HDR by over-expression of DNA repair proteins. To increase the efficiency of HDR, we will over-express human and nonhuman proteins involved in repair of DSBs by HDR, concomitantly with the expression of the CRISPR/Cas components. 4. Improving HDR by inhibition of non-homologous end-joining (NHEJ) repair. NHEJ and HDR compete for the repair of DSBs, and there is evidence that inhibition of NHEJ can enhance HDR. Thus, we will use pharmacological inhibitors of the NHEJ protein DNA-PK as well as a dominant negative version of 53BP1 to increase HDR yields in the EGFR model. 5. Analysis of off-target editing and DNA instability. We will sequence likely off-target sites in the modified cells to analyze whether the protocols developed in this project lead to an increase in off-target editing. Genome instability will be analyzed by karyotyping. IMPACT This research program will significantly improve our knowledge of the determinants for successful HDR. It will lead to novel tools to help move genome modification from basic genetics toward practical applications in agriculture and human health.