A Safe Harbor-Targeted CRISPR/Cas9 Homology Independent Targeted Integration (HITI) System for Multi-Modality Reporter Gene-Based Cell Tracking

Imaging reporter genes can provide valuable, longitudinal information on the biodistribution, growth and survival of engineered cells in preclinical models and patients. A translational bottleneck to using reporter genes in patients is the necessity to engineer cells with randomly-integrating vectors. CRISPR/Cas9 targeted knock-in of reporter genes at a genomic safe harbor locus such as adeno-associated virus integration site 1 (AAVS1) may overcome these safety concerns. Here, we built Homology Independent Targeted Integration (HITI) CRISPR/Cas9 minicircle donors for precise AAVS1-targeted simultaneous knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency at the AAVS1 site using HITI vectors compared to homology-directed repair donor vectors. Characterization of select HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significantly increased Oatp1a1-mediated uptake of the clinically-approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. As few as 106 Oatp1a1-expressing cells in a 50 {micro}l subcutaneous injection could be detected in vivo with contrast-enhanced MRI. Contrast-enhanced MRI also improved the conspicuity of both sub-cutaneous and metastatic Oatp1a1-expressing tumours prior to them being palpable or even readily visible on pre-contrast images. Our work demonstrates the first CRISPR/Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multi-modal longitudinal in vivo imaging of cells. This work lays the foundation for safer, non-viral reporter gene tracking of multiple cell types.