A Novel Mouse Model for the Detection of Genome Editing

Project Summary/Abstract CRISPR/Cas9 genome editing is rapidly developing into therapeutic applications. Therefore, it has become increasingly important that tools for pre-clinical testing of novel reagents provide the maximum benefit for researchers. One critical aspect of the use of CRISPR/Cas9 is the delivery of the molecular components of the editing machinery to the target tissue. Delivery reagents, such as viral vectors, lipid nanoparticles, conjugated ribonucleoproteins, and others, need to be tested for high efficiency editing of target tissues. At the same time, the reagents must also be evaluated for editing in non-targeted tissues and organs, to avoid unexpected biological consequences. Therefore, animal reporter models that detect genome editing events, are valuable tools for researchers developing new genome editing strategies. Current animal reporters, primarily mice, rely on the expression of a fluorescent or bioluminescent molecule in response to a genome editing event. One widely used model is the “Ai9” mouse. In this transgenic strain, a “double-hit” strategy is employed, where simultaneous CRISPR/Cas9 nuclease DNA cutting events, followed by Non-Homologous End Joining (NHEJ), excises an upstream stop cassette, allowing expression of a tdTomato fluorescent protein. This model is particularly useful, since the tdTomato protein produces robust fluorescence, allowing detection of very rare events. However, the model also has a pitfall. Many editing events, especially NHEJ that generates indels instead of excision, do not result in expression of tdTomato. Therefore, the actual number of editing events is undercounted. Therefore, we propose an alternative “single-hit” strategy to detect CRISPR/Cas9 editing in the Ai9 mouse. The Cre recombinase protein will also excise the stop cassette in the Ai9 mouse. However, when the Cre protein is fused to a derivative of the human estrogen receptor ligand binding domain called ERT2, it is retained in the cytoplasm, and remains inactive. We will design and test guide RNAs for CRISPR/Cas9 editing that will target this ERT2 domain. The resulting editing of the ERT2 is anticipated to disable the retention of the Cre recombinase in the cytoplasm, allowing the protein to translocate to the nuclease and excise the stop cassette. We predict that this strategy will be significantly more sensitive for the detection of editing events. In addition, this method will be adaptable to other Cre-inducible systems with alternative fluorophores and bioluminescent proteins as reporters. Finally, we expect that this strategy can be used with other CRISPR/Cas9-derived activities, notably base editing.