Development and Evaluation of A CRISPR-based Diagnostic For 2019-novel Coronavirus

BackgroundThe recent outbreak of infections by the 2019 novel coronavirus (2019-nCoV), the third zoonotic CoV has raised great public health concern. The demand for rapid and accurate diagnosis of this novel pathogen brought significant clinical and technological challenges. Currently, metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) are the most widely used molecular diagnostics for 2019-nCoV. Methods2019-nCoV infections were confirmed in 52 specimens by mNGS. Genomic information was analyzed and used for the design and development of an isothermal, CRISPR-based diagnostic for the novel virus. The diagnostic performance of CRISPR-nCoV was assessed and also compared across three technology platforms (mNGS, RT-PCR and CRISPR) Results2019-nCoVs sequenced in our study were conserved with the Wuhan strain, and shared certain genetic similarity with SARS-CoV. A high degree of variation in the level of viral RNA was observed in clinical specimens. CRISPR-nCoV demonstrated a near single-copy sensitivity and great clinical sensitivity with a shorter turn-around time than RT-PCR. ConclusionCRISPR-nCoV presents as a promising diagnostic option for the emerging pathogen.

Possible Involvement of F1F0-ATP synthase and Intracellular ATP in Keratinocyte Differentiation in normal skin and skin lesions.

The F1F0-ATP synthase, an enzyme complex, is mainly located on the mitochondrial inner membrane or sometimes cytomembrane to generate or hydrolyze ATP, play a role in cell proliferation. This study focused on the role of F1F0-ATP synthase in keratinocyte differentiation, and its relationship with intracellular and extracellular ATP (InATP and ExATP). The F1F0-ATP synthase ß subunit (ATP5B) expression in various skin tissues and confluence-dependent HaCaT differentiation models was detected. ATP5B expression increased with keratinocyte and HaCaT cell differentiation in normal skin, some epidermis hyper-proliferative diseases, squamous cell carcinoma, and the HaCaT cell differentiation model. The impact of InATP and ExATP content on HaCaT differentiation was reflected by the expression of the differentiation marker involucrin. Inhibition of F1F0-ATP synthase blocked HaCaT cell differentiation, which was associated with a decrease of InATP content, but not with changes of ExATP. Our results revealed that F1F0-ATP synthase expression is associated with the process of keratinocyte differentiation which may possibly be related to InATP synthesis.