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

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.

Endocytic recycling is central to circadian collagen fibrillogenesis and disrupted in fibrosis

“…Using this system, a GFP signal will only be present when VPS33b interacts with collagen-I. We, and others, have previously demonstrated that insertion of tags at the N-terminal of pro α 2(I) or pro α 1(I) chain does not interfere with collagen-I folding or secretion 29, 30 . To determine which terminus of the VPS33b protein GFP1-10 should be added, we inserted BFP on either the N-terminal (VPSnBFP) and C-terminal (VPScBFP) ends of VPS33b and imaged the cells.…”

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells

“…To monitor the replication of SARS-CoV-2 we generated DNA sequences that encode the SARS-CoV-2 variant Wuhan-Hu-1 (NC_045512.2, wild-type) and a modified traceable virus where Orf7a is replaced with the sequence encoding NanoLuciferase (SARS-Cov-2-ΔOrf7a-NLuc, Figure 1A ). NanoLuciferase (NLuc) is an enzyme that produces light when supplied with its substrate (coelenterazine) and is readily detectable even at low quantities 2 . Orf7a has been removed in SARS-CoV and SARS-CoV-2 and yielded infectious viral clones 4–6 , it is not highly conserved in beta-coronaviruses, and several disrupting mutations of Orf7a of the non-structural protein are known 7 .…”

“…1A). NanoLuciferase (NLuc) is an enzyme that produces light when supplied with its substrate (coelenterazine) and is readily detectable even at low quantities (Calverley et al, 2020). Orf7a has previously been removed in SARS-CoV and SARS-CoV-2 and yielded infectious and replicative virus particles (Thi Nhu Thao et al, 2020;Xie et al, 2020a;Xie et al, 2020b).…”

“…In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID-19.The Food and Drug Administration (FDA) and the European Medicines Agency (EMA) work with pharmaceutical companies to develop safe and effective drugs for the benefit of public health. Using a library of 1971 FDA-approved compounds, here we used a traceable clone of SARS-Cov-2 to identify well-characterized drugs that could potentially slow the infection and replication of the SARS-CoV-2 virus in human cells.We had previously developed an approach to quantitate collagen synthesis by using CRISPR-Cas9 to insert the gene encoding NanoLuciferase (NLuc) into the Col1a2 gene in fibroblasts (Calverley et al, 2020). Here, we adapted the approach to tag the SARS-CoV-2 virus with NLuc, and then used the recombinant NLuc-tagged virus to monitor virus replication and to identify drugs that slow the replication process.…”

“…We had previously developed an approach to quantitate collagen synthesis by using CRISPR-Cas9 to insert the gene encoding NanoLuciferase (NLuc) into the Col1a2 gene in fibroblasts (Calverley et al, 2020). Here, we adapted the approach to tag the SARS-CoV-2 virus with NLuc, and then used the recombinant NLuc-tagged virus to monitor virus replication and to identify drugs that slow the replication process.…”