Development of dual mode visualization of dCas9/fluorescent protein sensors in tumor xenografts on mice using optical clearing

Abstract

CRISPR/Cas9 system have been implemented in live imaging of edited or targeted genomic loci since it has been discovered. Catalytically inactive Cas9 (dCas9) has multiple applications with the most useful being the activation/repression of transcription. The use of dCas9 may assist in mapping of genes within chromatin structure at the level of individual cells and intact tissue.
The rationale for this research was to develop a set of tools for Cas9-mediated genome visualization at the whole-body level using fluorescence lifetime imaging based on dCas9 orthologs fused with fluorescent proteins (FP) as well as MRI of perfusion of tumor to co-localized it with fluorescence imaging for perspective dual mode visualization.
The visualization of dCas9-FP construct expression in tumor xenografts were fulfilled using planar imaging and FLIM followed by in vivo magnetic resonance imaging. To improve the contrast, a 0.7 M solution of gadobutrol was found to be useful for optical clearing of the skin surface resulting in overall increase of FI. I.V. injection of gadobutrol assisted in detection of regional tumor perfusion which delineated areas with inducible dCas9-FP chimera expression.
The application of optical clearing technique in vivo enabled high-contrast imaging of FP expression in tumor xenografts, which was combined with 3D GRE MRI assisted with a standard clinical MRI contrast agent for multi-modality mapping tumor perfusion and tumor fluorescence.

This work is financed by Russian Science Foundation Grant no. 22-14-00205. Optical clearing methods are developed under the Ministry of Science and Higher Education financing.

Speaker

Victoria Zherdeva
Bach Institute of Biochemistry, Research Center of Biotechnology of the RAS, Vavilov Institute of General Genetics,
Russia

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