Safe harbor-targeted CRISPR-Cas9 homology-independent targeted integration for multimodality reporter gene-based cell tracking
John J. Kelly, Moe Saee-Marand, Nivin N. Nyström, Melissa M Evans, Yuanxin Chen, Francisco M. Martínez, Amanda M. Hamilton, John A. Ronald
Abstract
) 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.