Evaluation of a lyophilized CRISPR-Cas12 assay for a sensitive, specific, and rapid detection of SARS-CoV-2

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55–100) positive samples and 104/105 (99.05%; 95% CI: 94.81–99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18–99.88) positive samples and 9/9 (100%; 95% CI: 66.37–100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45–36.90) and dilutions of heat-inactivated virus (range: 2.5–100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.Instituto de VirologíaFil: Curti, Lucía Ana. CASPR Biotech; Estados UnidosFil: Primost, Ivana. Hospital Municipal de Trauma y Emergencias Dr. Federico Abete. Genetics and Molecular Biology Laboratory; ArgentinaFil: Valla, Sofia. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires (CITNOBA). Centro de Investigaciones Básicas y Aplicadas (CIBA); ArgentinaFil: Valla, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ibañez Alegre, Daiana. Universidad Nacional de Misiones. Instituto de Biología Subtropical. Laboratorio Grupo de Investigación en Genética Aplicada (GIGA); ArgentinaFil: Ibañez Alegre, Daiana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olguin Perglione, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Olguin Perglione, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Repizo, Guillermo Daniel. CASPR Biotech; Estados UnidosFil: Lara, Julia. CASPR Biotech; Estados UnidosFil: Parcerisa, Ivana. CASPR Biotech; Estados UnidosFil: Palacios, Antonela. CASPR Biotech; Estados UnidosFil: Llases, María Eugenia. CASPR Biotech; Estados UnidosFil: Rinflerch, Adriana. Universidad Nacional de Misiones. Instituto de Biología Subtropical. Laboratorio Grupo de Investigación en Genética Aplicada (GIGA); ArgentinaFil: Rinflerch, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barrios, Melanie. Universidad de Buenos Aires. Instituto de Producción Agropecuaria; ArgentinaFil: Pereyra Bonnet, Federico. CASPR Biotech; Estados UnidosFil: Gimenez, Carla Alejandra. CASPR Biotech; Estados UnidosFil: Marcone, Débora Natalia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología, Biotecnología y Genética. Cátedra de Virología; ArgentinaFil: Marcone, Débora Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Estudio bioquímico y estructural del ensamblado del centro CuA de Arabidopsis thaliana

El cobre es un metal de transición clave para la vida, dado que permite a las proteínas catalizar una variedad de reacciones esenciales. El centro de cobre “CuA” de la Citocromo c Oxidasa (COX) es esencial para la respiración aeróbica de los organismos dependientes de COX. Una serie de genes que codifican para factores de ensamblado de dicho centro, son esenciales para la actividad de COX. Entre ellos, las proteínas de la familia Sco se encuentran altamente conservadas, y se ha demostrado que cumplen funciones variables entre organismos. Las plantas expresan dos proteínas de la familia Sco, Hcc1 y Hcc2, siendo Hcc1 esencial para la viabilidad de la planta. En el presente tesis se diseñó una proteína quimérica que funciona como modelo soluble de la subunidad CoxII de Arabidopsis thaliana, que contiene al centro CuA. Se analizaron sus características estructurales mediante varias espectroscopías, y se resolvió su estructura cristalográfica. Se identificaron y analizaron perturbaciones en la estructura electrónica del centro de cobre, causadas por mutaciones en la segunda esfera de coordinación. Se identificó la presencia de densidad electrónica sobre un ligando clave del centro CuA, la metionina axial. Esta información valida la participación de dicho residuo en la vía de trasferencia electrónica de la COX. Además, se expresó el dominio soluble del proteína Hcc1 de A. thaliana. Se confirmó que es capaz de unir cobre con elevada afinidad, se analizó la estructura del sitio de cobre en sus dos estados de oxidación posibles y se resolvió su estructura cristalográfica. Además, analizó su función in-vitro, demostrando que esta proteína es una metalochaperona, capaz de insertar iones de cobre específicamente para ensamblar el centro CuA.Fil: Llases, María Eugenia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina

Biochemistry of copper site assembly in heme-copper oxidases: a theme with variations

Copper is an essential cofactor for aerobic respiration, since it is required as a redox cofactor in Cytochrome c Oxidase (COX). This ancient and highly conserved enzymatic complex from the family of heme-copper oxidase possesses two copper sites: CuA and CuB. Biosynthesis of the oxidase is a complex, stepwise process that requires a high number of assembly factors. In this review, we summarize the state-of-the-art in the assembly of COX, with special emphasis in the assembly of copper sites. Assembly of the CuA site is better understood, being at the same time highly variable among organisms. We also discuss the current challenges that prevent the full comprehension of the mechanisms of assembly and the pending issues in the field.Para citar este articulo: Llases, M.-E.; Morgada, M.N.; Vila, A.J. Biochemistry of Copper Site Assembly in Heme-Copper Oxidases: A Theme with Variations. Int. J. Mol. Sci. 2019, 20, 3830. https://doi.org/10.3390/ijms20153830Fil: Lllases, María Eugenia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Morgada, Marcos N. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Morgada, Marcos N. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Vila, Alejandro J. Plataforma de Biología Estructural y Metabolómica (PLABEM); Argentina.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina

Arabidopsis thaliana Hcc1 is a Sco‐like metallochaperone for Cu A assembly in Cytochrome c Oxidase

International audienceThe assembly of the CuA site in Cytochrome c Oxidase (COX) is a critical step for aerobic respiration in COX-dependent organisms. Several gene products have been associated with the assembly of this copper site, the most conserved of them belonging to the Sco family of proteins, which have been shown to perform different roles in different organisms. Plants express two orthologs of Sco proteins: Hcc1 and Hcc2. Hcc1 is known to be essential for plant development and for COX maturation, but its precise function has not been addressed until now. Here, we report the biochemical, structural and functional characterization of Arabidopsis thaliana Hcc1 protein (here renamed Sco1). We solved the crystal structure of the Cu+1 -bound soluble domain of this protein, revealing a tri coordinated environment involving a CxxxCxn H motif. We show that AtSco1 is able to work as a copper metallochaperone, inserting two Cu+1 ions into the CuA site in a model of CoxII. We also show that AtSco1 does not act as a thiol-disulfide oxido-reductase. Overall, this information sheds new light on the biochemistry of Sco proteins, highlighting the diversity of functions among them despite their high structural similarities. DATABASE: PDB entry 6N5U (Crystal structure of Arabidopsis thaliana ScoI with copper bound)

Unexpected electron spin density on the axial methionine ligand in Cu A suggests its involvement in electron pathways

International audienceThe CuA center is a paradigm for the study of long-range biological electron transfer. This metal center is an essential cofactor for terminal oxidases like cytochrome c oxidase, the enzymatic complex responsible for cellular respiration in eukaryotes and in most bacteria. CuA acts as an electron hub by transferring electrons from reduced cytochrome c to the catalytic site of the enzyme where dioxygen reduction takes place. Different electron transfer pathways have been proposed involving a weak axial methionine ligand residue, conserved in all CuA sites. This hypothesis has been challenged by theoretical calculations indicating the lack of electron spin density in this ligand. Here we report an NMR study with selectively labeled methionine in a native CuA. NMR spectroscopy discloses the presence of net electron spin density in the methionine axial ligand in the two alternative ground states of this metal center. Similar spin delocalization observed on two second sphere mutants further supports this evidence. These data provide a novel view of the electronic structure of CuA centers and support previously neglected electron transfer pathways

Arabidopsis thaliana Hcc1 is a Sco-like metallochaperone for Cu A assembly in Cytochrome c Oxidase

The assembly of the CuA site in Cytochrome c Oxidase (COX) is a critical step for aerobic respiration in COX-dependent organisms. Several gene products have been associated with the assembly of this copper site, the most conserved of them belonging to the Sco family of proteins, which have been shown to perform different roles in different organisms. Plants express two orthologs of Sco proteins: Hcc1 and Hcc2. Hcc1 is known to be essential for plant development and for COX maturation, but its precise function has not been addressed until now. Here, we report the biochemical, structural and functional characterization of Arabidopsis thaliana Hcc1 protein (here renamed Sco1). We solved the crystal structure of the Cu+1-bound soluble domain of this protein, revealing a tri coordinated environment involving a CxxxCxnH motif. We show that AtSco1 is able to work as a copper metallochaperone, inserting two Cu+1 ions into the CuA site in a model of CoxII. We also show that AtSco1 does not act as a thiol-disulfide oxido-reductase. Overall, this information sheds new light on the biochemistry of Sco proteins, highlighting the diversity of functions among them despite their high structural similarities.Fil: Llases, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Lisa, María Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Plataforma de Biología Estructural y Metabolómica; ArgentinaFil: Morgada, Marcos Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Giannini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica; ArgentinaFil: Alzari, Pedro M. Université Paris Diderot - Paris 7; FranciaFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Unexpected Electron Spin Density on the Axial Methionine Ligand in CuA Suggests Its Involvement in Electron Pathways

The CuA center is a paradigm for the study of long-range biological electron transfer. This metal center is an essential cofactor for terminal oxidases like Cytochrome c oxidase, the enzymatic complex responsible for cellular respiration in eukaryotes and in most bacteria. CuA acts as an electron hub by transferring electrons from reduced cytochrome c to the catalytic site of the enzyme where dioxygen reduction takes place. Different electron transfer pathways have been proposed involving a weak axial methionine ligand residue, conserved in all CuA sites. This hypothesis has been challenged by theoretical calculations indicating the lack of electron spin density in this ligand. Here we report an NMR study with selectively labeled methionine in a native CuA. NMR spectroscopy discloses the presence of net electron spin density in the methionine axial ligand in the two alternative ground states of this metal center. Similar spin delocalization observed on two second sphere mutants further supports this evidence. These data provide a novel view of the electronic structure of CuA centers and support previously neglected electron transfer pathways. </p