Tet-Regulated Expression and Optical Clearing for In Vivo Visualization of Genetically Encoded Chimeric dCas9/Fluorescent Protein Probes

Abstract

The catalytically inactive mutant of Cas9 (dCas9) endonuclease has multiple biomedical applications, with the most useful being the activation/repression of transcription. dCas9 family members are also emerging as potential experimental tools for gene mapping at the level of individual live cells and intact tissue. We performed initial testing on a set of tools for Cas9-mediated visualization of nuclear compartments. We investigated doxycycline (Dox)-inducible (Tet-On) intracellular distribution of constructs encoding dCas9 orthologs from St. thermophilus (St) and N. meningitides (Nm) fused with EGFP and mCherry fluorescent proteins (FP) in human A549 cells. We also studied time-dependent expression of these chimeric fluorescent constructs (dCas9-FP) after Tet-On induction in live cells and compared it with the time course of dCas9-FP expression in experimental dCas9-FP-expressing tumor xenografts using a combination of fluorescence imaging and in vivo contrast-assisted magnetic resonance imaging for assessing the extent of tumor perfusion. In vivo Dox-induction of mCherry-chimera expression occurred in tumor xenografts as early as 24 h post-induction and was visualized by using optical clearing (OC) of the skin. OC via topical application of gadobutrol enabled high-contrast imaging of FP expression in tumor xenografts due to a 1.1–1.2-fold increase in FI in both the red and green channels.

Visualizing the Nucleome Using the CRISPR–Cas9 System: From in vitro to in vivo

“…Cas9 endonucleases and their catalytically inactive dCas9 mutants are able to form functional fusion proteins with FPs [ 5 , 127 , 211 ], base-editing enzymes (e.g., APOBEC deaminase) [ 212 ], and ascorbate peroxidase capable of biotinylating DNA-bound proteins [ 213 ]. However, the use of enzyme tags for obtaining fluorescent pairs of dCas9 orthologs for direct enzyme-mediated labeling with fluorescent substrates has not yet been investigated.…”