Genome editing in vivo requires safe and efficient delivery of reagents to a target tissue. At the same time, delivery to non-target tissues, especially germline cells, must be assessed for potential deleterious effects. Engineered reporter mouse strains allow both a qualitative and quantitative assessment of genome editing efficiency following delivery. At the GETC, our primary reporter mouse strains rely on the activation of fluorescent reporters in response to a successful editing event. Using high-throughput scanning to detect native fluorescence from the reporter, we can rapidly image a panel of organs from a mouse to identify edited cells in both target and non-target tissues. In many cases, coincident nuclei staining allows us to make tentative cell type identifications based on morphology.
Mouse Strains
The GETC maintains active colonies of several fluorescent reporter mouse models. We house the Ai9 and Ai6 strains widely used for in vivo assessment of CRISPR/Cas9 editing. In addition, we house Ai9 and Ai6 derivative strains that have been engineered for single-guide RNA sequence editing and homology-directed repair editing with CRISPR/Cas. Visit Resources for a full list. Alternative models are also from public repositories.
Delivery Vectors
Our center has extensive experience with multiple vectors for the targeted delivery of genome editing reagents. We have successfully tested viral vectors, including multiple natural and engineered adeno-associated virus (AAV) serotypes, as well as adenovirus. We have also successfully tested lipid nanoparticles, amphiphilic peptides, and virus-like particles as delivery reagents for genome editing. Established delivery routes include intravenous (both tail vein and retro-orbital), intraperitoneal, intranasal, and intratracheal, as well as oral gavage. Other routes of administration or delivery modalities are possible and evaluated on a case-by-case basis.
Testing Pipeline
Our standard genome editing testing pipeline in reporter mice begins with the application of the delivery reagent to a cohort of male and female reporter mice, followed by careful health monitoring of the animals. After enough time has passed to ensure maximum editing has occurred, individual organs are imaged and analyzed for the presence of fluorescent signal, indicating a successful editing event. Editing events in target tissues are quantified, while non-target tissues are carefully assessed for the presence of fluorescent cells indicating unexpected editing. Selected tissues are also examined for evidence of adverse effects from the delivery reagent.
We also offer additional services to adapt or expand the editing analysis. Flow cytometry is available as an alternative to image analysis for quantification in certain tissues. Immunostaining can be performed for cell type identification in edited tissues. In the absence of a suitable reporter mouse model, we can perform targeted sequencing (in collaboration with Rice University) to identify and quantify genome editing. This analysis can be expanded to pre-selected off-target editing sites in the genome, as well as non-target organs.
Proposing a Reporter Model Testing Experiment
To initiate preliminary discussions regarding a Reporter Model Testing experiment, please fill out the form at the link below or email us directly at GETC-info@bcm.edu. Our personnel will contact you to set up a discussion to match your in vivo editing requirements with our resources and capabilities.