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

2021 ◽  
Vol 7 (4) ◽  
pp. eabc3791
Author(s):  
John J. Kelly ◽  
Moe Saee-Marand ◽  
Nivin N. Nyström ◽  
Melissa M. Evans ◽  
Yuanxin Chen ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Cell Tracking ◽  
Reporter Genes ◽  
Safe Harbor ◽  
Growth And Survival ◽  
Homology Directed Repair ◽  
Reporter Activity ◽  
Contrast Enhanced ◽  
Targeted Integration

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.

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

2020 ◽  
Author(s):  
John J Kelly ◽  
Moe Saee-Marand ◽  
Nivin N Nyström ◽  
Yuanxin Chen ◽  
Melissa M Evans ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Cell Tracking ◽  
Integration Site ◽  
Reporter Genes ◽  
Safe Harbor ◽  
Enhanced Mri ◽  
Contrast Enhanced ◽  
Targeted Integration ◽  
Contrast Enhanced Mri

AbstractImaging 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 µl 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.

scholarly journals Contrast-Enhanced In Vivo Imaging of Breast and Prostate Cancer Cells by MRI

Cell Cycle ◽  
2005 ◽  
Vol 5 (1) ◽  
pp. 113-119 ◽  
Author(s):  
Olga Rodriguez ◽  
Stanley Fricke ◽  
Christopher Chien ◽  
Luis Dettin ◽  
John VanMeter ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Cells ◽  
In Vivo Imaging ◽  
Prostate Cancer Cells ◽  
Contrast Enhanced ◽  
Breast And Prostate Cancer

Ternary-Responsive Drug Delivery with Activatable Dual Mode Contrast-Enhanced in Vivo Imaging

2018 ◽  
Vol 10 (38) ◽  
pp. 31947-31958 ◽  
Author(s):  
Shuangshuang Ren ◽  
Jie Yang ◽  
Lan Ma ◽  
Xincong Li ◽  
Wenlei Wu ◽  
...  
Keyword(s):  
Drug Delivery ◽  
In Vivo Imaging ◽  
Dual Mode ◽  
Contrast Enhanced

scholarly journals Real-time detection of circulating tumor cells in living animals using functionalized large gold nanorods

2018 ◽  
Author(s):  
Rebecca Dutta ◽  
Orly Liba ◽  
Elliott D SoRelle ◽  
Yonatan Winetraub ◽  
Vishnu Ramani ◽  
...  
Keyword(s):  
Contrast Agents ◽  
Tumor Cells ◽  
Gold Nanorods ◽  
Cell Tracking ◽  
Speckle Noise ◽  
Speckle Reduction ◽  
Exciting Opportunity ◽  
Contrast Enhanced ◽  
The Individual

Optical coherence tomography (OCT) with significant speckle reduction can be used with highly-scattering contrast agents for noninvasive, contrast-enhanced imaging of living tissue at the cellular scale. The advantages of reduced speckle noise and improved targeted contrast can be harnessed to track objects as small as 2 μm in vivo, with the potential for cell tracking and counting in living subjects. Here we demonstrate the use of Large Gold Nanorods (LGNRs) as contrast agents for detecting individual micron-sized polystyrene beads and single myeloma cells in blood circulation using speckle modulating-OCT (SM-OCT). This is the first time that OCT has been used to image at the individual cell scale in vivo. This technical capability presents an exciting opportunity for the dynamic detection and quantification of tumor cells circulating in living subjects.

In-vivo imaging of carotid plaque neoangiogenesis with contrast-enhanced harmonic ultrasound

2009 ◽  
Vol 134 (3) ◽  
pp. e110-e112 ◽  
Author(s):  
Theodore G. Papaioannou ◽  
Manolis Vavuranakis ◽  
Aristides Androulakis ◽  
George Lazaros ◽  
Ioannis Kakadiaris ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Carotid Plaque ◽  
Contrast Enhanced
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