Developing Improved Reporter Mouse Models for In Vivo Gene Editing

About This Grant

PROJECT SUMMARY CRISPR-Cas9 technologies have revolutionized genetic engineering and have led to a renaissance in genetic therapy research. The initial CRISPR discovery was that of an RNA-guided endonuclease capable of creating double-strand DNA breaks which can be precisely corrected using DNA donor-mediated homology- directed repair (HDR). While an advance, this approach is complicated by low efficiency and non-therapeutic side products arising from non-homologous end joining (NHEJ). Among the most promising of various newer approaches are A- and C-base editing, which allow for precise genome modification of adenine and cytosine residues, respectively, without the generation (and drawbacks) of double-strand DNA breaks. Testing the safety and efficacy of such approaches relies on preclinical studies often performed using mouse models. To this point, mouse gene editing reporter strains serve many roles in characterizing gene editors. For example, gene editing reporter strains can be used to track the timing and location of gene editing, assess gene editing efficiency, optimize gene editor delivery, discriminate between repair mechanisms, and lay the groundwork for clinical studies through preclinical testing of emerging therapeutics. During Phase 1 of the Somatic Cell Genome Editing Consortium, The Jackson Laboratory established a Gene Editing and Testing Center to design, engineer, and deploy new reporter models. We created three new mouse gene editing reporter strains that can effectively indicate HDR, NHEJ, and A-base editing activity at single- cell resolution through the activation of a fluorescent marker. These models are now available for public distribution and are well-utilized by the therapeutic gene editing community. Despite these successes, gaps remain in the mouse gene editing and characterization toolbox limiting the number of editing outcomes that can be reliably measured, in vivo. This proposal aims to address these needs by constructing and characterizing two mouse gene editing reporter strains. The first strain will enable detection of C-base editor activity, an important editing technology with growing therapeutic interest thanks to the development of C-base editor variants with high specificity. The second strain is a multifunctional reporter that addresses the specific need for a reporter that can faithfully detect most NHEJ events, but that also reports, HDR, A-base editing, and Cre-directed recombination. In all, this multifunctional reporter assesses a number of important activities for evaluating new gene editing platforms. Each of these reporters will be functionally characterized across three levels of biological complexity, qualifying them for further use in preclinical testing.