Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging.

Ben C Calverley,Karl E Kadler,Adam Pickard

doi:10.3390/cells9092070

Abstract

The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation. Collagen was chosen because of the clinical interest in its dysregulation in cardiovascular and musculoskeletal disorders, and in fibrosis, which is a confounding factor in 45% of deaths, including those brought about by cancer. Collagen is also the cargo protein of choice for studying protein secretion because of its unusual shape and size. However, the use of overexpression promoters (which drowns out endogenous regulatory mechanisms) is often needed to achieve good signal/noise ratios in fluorescence microscopy of tagged collagen. We show that endogenous knock-in of NLuc, combined with its high brightness, negates the need to use exogenous promoters, preserves the circadian regulation of collagen synthesis and the responsiveness to TGF-β, and enables time-lapse microscopy of intracellular transport compartments containing procollagen cargo. In conclusion, we demonstrate the utility of CRISPR-Cas9-mediated endogenous NLuc tagging to robustly quantitate extracellular, intracellular, and subcellular protein levels and localisation.

Highlights

Quantitation of DNA and RNA is routine in research and diagnostic laboratories, and makes use of base pair hybridisation to ensure specificity and identification
We show that the light produced by NLuc is sufficiently bright to obtain dynamic quantitative information on the number of endogenous collagen-I molecules trafficking through living cells and being secreted and incorporated into the extracellular matrix
Mouse NIH3T3 is a mouse embryonic fibroblast that has been immortalised by a standardised passaging procedure [20]. These fibroblasts produce both proα1(I) and proα2(I), which make up the heterotrimeric type I procollagen molecule, and assemble the type I collagen into fibrils, making them suitable for studying the entire biosynthesis and fibril assembly processes

Summary

Introduction

Quantitation of DNA and RNA is routine in research and diagnostic laboratories, and makes use of base pair hybridisation to ensure specificity and identification. Similar approaches are not available for proteins Methods such as ELISA immunoassays and western blotting are widely used to estimate levels of proteins, but spatial resolution is lost, and they are unsuitable for live cell studies where dynamic readouts are required. In this regard, the use of fluorescent proteins and chemical tags has revolutionised cell biology, but quantitation through fluorescence is not without technical difficulties associated with quenching, extensive wash-out, and the influence of the local environment on the fluorescence signal. Bioluminescence produced when luciferase hydrolyses luciferin-based substrates offers a practical alternative to using fluorescent tags. When tagged to a protein of interest, luciferase emits visible

Methods

Results

Conclusion