Safe harbor-targeted CRISPR-Cas9 homology-independent targeted integration for multimodality reporter gene-based cell tracking

Abstract

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.

Sensitive Spatiotemporal Tracking of Spontaneous Metastasis in Deep Tissues via a Genetically-Encoded Magnetic Resonance Imaging Reporter

“…No significant difference was observed between R 1 at the 60-minute (5.03 ±0.28 Hz) and 90-minute timepoints (5.67 ± 0.32 Hz; n=3, p=0.053) as the first derivative approaches zero, suggesting that the intracellular Gd(III) concentration detected via magnetic resonance approaches a steady state ( R 1,max = 5.85 Hz) at about 90 min of incubation (96.9 ± 5.5% of R 1,max ; Figure 2d ). The slow uptake kinetics observed here as well as the slow cellular efflux of the probe ( Supplementary Figure S4 ) reinforces the approach of previous studies utilizing oatp1a1 , wherein signal-to-background in mice reached a maximum at approximately 5 h post Gd-EOB-DTPA administration 23,25 .…”

“…Accordingly, our primary objective was to increase the sensitivity of the oatp1-MRI system for detection and dynamic tracking of oatp1-engineered cancer cells in a spontaneous metastasis model of breast cancer. We report that oatp1-MRI produces highly-sensitive, 3-dimensional, and high-resolution images of metastatic progression in live mice over time; we improve system sensitivity by three orders of magnitude relative to previous studies [23][24][25] , enabling detection of fewer than 10 3 cells per lesion, or 10 2 cells per voxel, in lung tissue. Importantly, oatp1-MRI enables detection of reporter gene signals that are unaffected by tissue depth or the presence of adjacent lesions.…”

“…For example, organic anion-transporting polypeptide 1 ( oatp1 ), which encodes a 12-transmembrane-domain integral membrane protein, was previously established as a reporter gene based on its ability to bind and transport gadolinium ethoxybenzyl diethylene triamine pentaacetic acid (Gd-EOB-DTPA) from the extracellular environment into oatp1 -engineered cells 23 . We and others applied oatp1 -MRI to in vivo cancer imaging and showcased its ability to generate detailed, high-resolution images of primary tumor architecture in mice 24 , but measured its in vivo detection limit at 10 6 cells per lesion 25 . Meanwhile, non-MR reporter genes recently achieved detection of single isolated cells on BLI 26 and detection of lesions on the order of 10 4 cells on positron emission tomography (PET) 27 .…”

Cell-derived extracellular vesicles for CRISPR/Cas9 delivery: engineering strategies for cargo packaging and loading

“…37 Cas9 MC DNA delivery by MVs constitutes a safe vector for effective gene editing in the clinic. 38 Lin and co-workers reported an exosome–liposome hybrid system developed by simple incubation to improve the loading capacity and delivery efficiency of exosomes. Unlike native exosomes, hybrid exosome particles can efficiently encapsulate CRISPR/Cas9 expression plasmids.…”

Molecular imaging of cellular immunotherapies in experimental and therapeutic settings

“…The use of the rat-derived organic anion transport polypeptide (OATP) 1A1 and human-derived OATP1B3 have also been investigated by Dr. Kevin Brindle’s group [ 72 ] as well as our group [ 73 , 74 ] as MRI reporter genes due to their ability to uptake a clinical gadolinium-based contrast agent. This reporter gene system has been used for cancer cell tracking in various mouse models, and ongoing work is focused on evaluating this reporter system’s clinically relevant cell types.…”