Imaging reporter genes provides longitudinal information on the biodistribution, growth, and survival of engineered cells in vivo. A translational bottleneck to using reporter genes is the necessity to engineer cells with randomly integrating vectors. Here, we built homology-independent targeted integration (HITI) CRISPR-Cas9 minicircle donors for precise safe harbor-targeted knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency using HITI vectors compared to homology-directed repair vectors. HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significant Oatp1a1-mediated uptake of the clinically approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. Contrast-enhanced MRI improved the conspicuity of both subcutaneous and metastatic Oatp1a1-expressing tumors before they became palpable or even readily visible on precontrast images. Our work demonstrates the first CRISPR-Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multimodal longitudinal in vivo imaging of cells.
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